Method and apparatus for acoustically controlling liquid solutions in microfluidic devices

ABSTRACT

Acoustic energy is used to control motion in a fluid. According to one embodiment, the invention directs acoustic energy at selected naturally occurring nucleation features to control motion in the fluid. In another embodiment, the invention provides focussed or unfocussed acoustic energy to selectively placed nucleation features to control fluid motion. According to one embodiment, the invention includes an acoustic source, a controller for controlling operation of the acoustic source, and one or more nucleation features located proximate to or in the fluid to be controlled.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/812,723, filed Mar. 20, 2001 now U.S. Pat. No. 6,948,843, whichclaims the benefit of and priority to U.S. Provisional PatentApplications 60/246,838, filed on Nov. 8, 2000, 60/198,923, filed Apr.21, 2000, and 60/191,297, filed on Mar. 21, 2000; and is acontinuation-in-part of International Patent Application PCT/US99/25274,filed on Oct. 28, 1999, which itself claims priority to U.S. ProvisionalPatent Applications 60/105,933, filed on Oct. 28, 1998; 60/110,460,filed on Dec. 1, 1998; 60/119,500, filed on Feb. 10, 1999; 60/143,440,filed on Jul. 13, 1999; and 60/148,279, filed on Aug. 11, 1999, thedisclosures of all of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention generally relates to non-contact mixing. Moreparticularly, in one embodiment, the invention is directed to a deviceand related methods for non-contact mixing and fluid control.

BACKGROUND OF THE INVENTION

Microfluidic devices, including “biochip” arrays, “laboratories on achip”, ultraminiaturized instruments, and the like, have become widelyused in research, development, and testing (including diagnostics).Examples include the study of biological-based processes, such asfunctional genomics (“DNA microarrays”), proteomics, and the like. Oftenthe underlying principle of these reaction devices is an initial bindingevent between material on a substrate within the device and material ina solution that is exposed to the substrate. Binding events are oftendiffusion limited and can be enhanced by mixing. Pre- andpost-processing such as washing and elution steps can also benefit frommixing in the device. Procedures not necessarily requiring binding, suchas electrophoresis and some types of chromatography, are also beingimplemented on very small devices, often using integrated circuittechnology from microelectronics processing. These and other processareas that have been or may be implemented in microfluidic deviceformats and which may benefit from mixing or enhanced fluid flow includeextraction, resuspension, solvation, emulsification, separation, anddetection.

It is difficult to actively mix or control fluid flow in small,microfluidic devices. These devices typically have internal dimensionsless than about 50 millimeters and flow velocities typically less thanabout 10 millimeters per second. For these devices, the Reynolds Numbersencountered are typically less than about one (1), so that flow issmooth and non -turbulent. Viscous laminar flow effects dominate andthere are no significant inertial effects. Under these conditions, flowstreamlines are parallel. In this domain, mass transfer or exchangeacross the streamlines typically occurs by diffusion. The detrimentaleffects of having a diffusion-based system on commercial products arenumerous, such as but not limited to a constraint on reducing assaytimes and difficulty in actively controlling intra-assay precision andaccuracy.

It is known that acoustic energy, particularly ultrasonic energy, may beused to effect mixing by multiple processes, including temperature,cavitation, and acoustic streaming. For example, acoustic-based mixinghas been shown to improve antibody detection and reduce incubationtimes. However, in the prior art, ultrasonic mixing is performed with anonfocused transducer operating in the 20,000 to 40,000 Hz range. Thetransducer contacts the sample fluid directly, which severely limitspractical applications, particularly with microfluidic devices.Moreover, when cavitation bubbles formed in older devices collapse, thebubble nucleation, growth and collapse is not directed, nor controlleddevice.

SUMMARY OF THE INVENTION

To solve the above discussed deficiencies in prior art approaches, inone embodiment, the invention provides a new apparatus that improvesprocesses related to microfluidic devices and similar structures,including biochips, lab-on-a-chip devices, and multi-well plate formats.In a further embodiment, the invention also provides for treatment ofother internal spaces of microfabricated devices having cavitationpromoting features or textures.

In one aspect, the apparatus of the invention controls fluid flow byproviding nucleation features at particular locations to lower acavitation threshold. In another aspect, the apparatus of the inventioncontrols an acoustic field to reduce the cavitation threshold atpre-existing cavitation features. The invention may control the acousticfield, for example, through focussing, blocking, and/or reflectingtechniques.

According to one embodiment, the invention provides an apparatus andrelated methods for its use in mixing and fluid movement control. In oneaspect, the apparatus includes an acoustic energy source, such as anultrasound transducer; a controller for providing a waveform type andamplitude controlling signal to the transducer; and one or morenucleation promoting features. The nucleation features may be, forexample, mechanical, electrical or chemical in nature. The apparatus mayincorporate feedback control mechanisms for adjusting characteristics ofthe acoustic field generated by the acoustic source.

In one embodiment, the volume of fluid mixed or controlled is betweenabout 1 pico liter (pl) to about 3 ml. In another embodiment, the volumeof fluid is between about 10 nanoliters (nl) to about 100 nl

In some embodiments, the acoustic source/transducer, the controller andthe nucleation promoting features are fabricated integrally with amicrodevice containing a fluid to be mixed or caused to flow. In otherembodiments one or both of the acoustic source and the controller arefabricated separately from and located remotely to the microdevice. Inembodiments where the acoustic source is external to or separate fromthe microdevice, the acoustic source couples to the microdevice, forexample, by way of a liquid, gel solid, vapor or gas. In alternateembodiments, the acoustic source may be in contact with the fluid to becontrolled. In other embodiments, a portion of a microdevice, such as awall or other structure, is the couplant that couples acoustic energyfrom an acoustic source to the fluid to be controlled.

The acoustic energy source may be any suitable source, such as apiezoelectric acoustic source. The source may or may not be focussed.Source frequencies in the range of about 10 kilohertz (kHz) to about 100megahertz (MHz) are preferred in the practice of the invention, becauseat these frequencies, the acoustic field may be usefully focussed orotherwise shaped and controlled. For a focussed transducer, theresulting focal zone in the microdevice can be small. The size of thefocal zone varies approximately inverse to the frequency. By way ofexample, at about 3 MHz, focal zones of about 1 mm in diameter and about4 mm long can be obtained. By way of further example, at about 10 MHz,the diameter can be less than about 0.3 mm and the length about 2 mm.Non-focussed transducers operating at these frequencies may have usefulnatural focussing. For example, a non-focussed transducer 25 mm indiameter operating at 1 MHz will produce an acoustic beam having anatural focal zone about 7 mm in diameter at its narrowest point. Thesesizes may be large relative to the regions of interest inmicrofabricated devices. Preferably, the ultrasonic energy delivered issufficiently intense to at least form a cavitation bubble in a targetzone. In a preferred embodiment, the ultrasonic energy delivered issufficiently intense to oscillate the bubble in the target zone. Atstill higher energies the delivered ultrasonic energy can result information and streaming of bubble(s) in the target zone; this ispreferred for some types of microfluidic devices.

In one embodiment, a piezoelectric acoustic transducer is integrallyformed in a microfabricated device In a further integrated embodiment,an array of piezoelectric acoustic drivers are co-fabricated with anarray of active sites adapted, for example, for detection or reaction.An example of a suitable piezoelectric acoustic source is provided inthe fabrication of atomic force microscopes and similar devices.

In some embodiments, the acoustic source is movable with respect to thetarget microdevice components. In other embodiments, the targetmicrodevice is moveable with respect to the acoustic source. In anotherembodiment, the invention provides an individual pathway from theacoustic source to each element of an array in the microdevicecontaining the fluid to be controlled. According to one embodiment, thisis accomplished by providing an acoustic waveguide to conduct acousticenergy to each element of the array of the microdevice.

Also, as noted, a sound conducting material, such as water, vapor, gas,gel, or solid material, can be placed between the acoustic source andthe microdevice. For example, a water bath may be employed to conductacoustic energy from the acoustic source of the mixing apparatus to amicrodevice containing the liquid to be mixed.

In one embodiment, nucleation sites are positioned at specificlocations. The ultrasonic energy is directed to a region containingnucleation sites. In one embodiment, the nucleation sites are featuresor textures that act to promote the formation of bubbles and gascavities within a fluid. The features may be point features such aspits, crevices, defects or linear features such as scratches, grooves orridges, or arrays of point features or linear features. The features mayalso be embodied in variations in hydrophobicity, wetability, surfaceenergy and/or a distribution of impurities or contaminants on or in asurface of the microdevice. Multiple features may be employed in regulararrays or randomly in a region to create cavitation inducing textureswithin a microdevice. The features are disposed at locations on orwithin a fluid device such that desired mixing or flow patterns areachieved in the presence of an appropriate acoustic field. Thenucleation sites may also be disposed in the liquid to be controlled.Such nucleation sites can be, for example, a particle, bead, microsphereof resin.

Localized variations in material properties, such as acoustic impedance,hydrophobicity, wetability, or surface energy may be employed asnucleation sites to create cavitation inducing loci on a surface of themicrodevice and may be beneficially combined with the above discussednucleation sites. A distribution of impurities in or on a surface of themicrodevice may also be employed as nucleation-promoting features. Also,electrodes can be employed to facilitate nucleation at particular sites.

In a further embodiment, the invention directs acoustic energy tocommonly occurring irregularities, which may be created in theconstruction of fabricated fluid devices, such as rough cut edges, tonucleate bubbles at predictable, reliable locations.

In one aspect of the invention, cavitation effects may be used to inducerotational or convective flow within a fluid in a microdevice chamber,causing local mixing. In another aspect of the invention, cavitationeffects may be used to induce bubble formation and decay within adefined portion of a fluid conduit, thereby inducing a localized valveeffect. In another aspect of the invention, bubbles or cavities aregenerated and released from a nucleation locus or loci such that theystream in response to acoustic field gradients for the purpose ofcausing fluid flow within a chamber or conduit.

The above effects may be applied to a variety of fluids, particularlybiochemical fluids, molecules and reactions. The localized acousticenergy may be used for a variety of purposes. Among these are mixingfluids, moving fluids, improving reaction rates, accelerating molecularinteractions, conditioning reaction sites, denaturing molecules, and ifrequired, providing local heating.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and objects of the invention,reference should be made to the following detailed description and theaccompanying drawings, in which the drawings are not necessarily toscale, and in which:

FIG. 1 is a schematic illustration of one embodiment of the apparatusaccording to the invention;

FIG. 2 is a schematic illustration of one example of sonic energycontrol showing sine waves at a variable amplitude and frequency;

FIG. 3 is a schematic illustration of one example of an intra-samplepositioning (dithering) profile showing height, height step, and radius;

FIG. 4A is a schematic illustration of a vertical-sided treatmentvessel;

FIG. 4B is a schematic illustration of a conical treatment vessel;

FIG. 4C is a schematic illustration of a curved treatment vessel;

FIGS. 5A-5C are schematic illustrations of several embodiments of atreatment vessel with a combination of an upper and lower member andsamples in the vessels prior to treatment;

FIG. 6A is a schematic illustration of a treatment vessel positionedover a collection container prior to transferring the contents of thevessel to the container;

FIG. 6B is a schematic illustration of a treatment vessel positionedover a collection container after transferring some of the contents ofthe vessel to the container;

FIG. 7 is a schematic illustration of an embodiment of the inventionwith a microtiter plate containing samples, such that one of the wellsof the microtiter plate is positioned at the focus point of sonicenergy;

FIG. 8 is a conceptual diagram detailing the life cycle of a cavitationbubble formation and collapse, according to an illustrative embodimentof the invention;

FIG. 9 is a conceptual diagram showing the stages of nucleation andgrowth of a gas body in a microcavity in a substrate, according to anillustrative embodiment of the invention;

FIG. 10 is conceptual diagram depicting an acoustic microstreaming-basedmixing device according to an illustrative embodiment of the invention;

FIG. 11 is a conceptual diagram of an exemplary configuration of acavitation promoter array according to an illustrative embodiment of theinvention.

FIGS. 12A and 12B are conceptual diagrams depicting micro flow valveaccording to an illustrative embodiment of the invention;

FIG. 13 is a conceptual diagram depicting the directional accelerationof fluid flow according to an illustrative embodiment of the invention;

FIGS. 14A-14D are conceptual diagrams depicting mass transfer across afield of nucleation sites according to an illustrative embodiment of theinvention;

FIG. 15 is a conceptual diagram depicting mixing of fluid on amicroscope slide, according to an illustrative embodiment of theinvention;

FIGS. 16A-16B are conceptual diagrams depicting electrode cleaningaccording to an illustrative embodiment of the invention;

FIG. 17 is a conceptual diagram depicting an electrode nucleationfeature according to an illustrative embodiment of the invention; and

FIG. 18 is a conceptual diagram of a MEM constructed according to anillustrative embodiment of the invention.

DESCRIPTION OF AN ILLUSTRATIVE EMBODIMENT

“Acoustic energy” as used herein is intended to encompass such terms assonic energy, acoustic waves, acoustic pulses, ultrasonic energy,ultrasonic waves, ultrasound, shock waves, sound energy, sound waves,sonic pulses, pulses, waves, or any other grammatical form of theseterms, as well as any other type of energy that has similarcharacteristics to acoustic energy. “Focal zone” or “focal point” asused herein means an area where acoustic energy converges and/orimpinges on a target, although that area of convergence is notnecessarily a single focused point. As used herein, the terms“microplate,” “microtiter plate,” “microwell plate,” and othergrammatical forms of these terms can mean a plate that includes one ormore wells into which samples may be deposited. As used herein,“nonlinear acoustics” can mean lack of proportionality between input andoutput. For example, in our application, as the amplitude applied to thetransducer increases, the sinusoidal output loses proportionality suchthat eventually the peak positive pressure in the acoustic fieldincreases at a higher rate than the peak negative pressure. Also, waterbecomes nonlinear at high intensities, and in a converging acousticfield, the waves become more disturbed as the intensity increases towardthe focal point. Nonlinear acoustic properties of tissue can be usefulin diagnostic and therapeutic applications. As used herein, “acousticstreaming” means generation of fluid flow by acoustic waves. The effectcan be non-linear. Bulk fluid flow of a liquid in the direction of thesound field can be created as a result of momentum absorbed from theacoustic field. As used herein, “acoustic microstreaming” meanstime-independent circulation that occurs only in a small region of thefluid around a source or obstacle for example, an acoustically drivenbubble in a sound field. As used herein, “acoustic absorption” refers toa characteristic of a material relating to the material's ability toconvert acoustic energy into thermal energy. As used herein, “acousticimpedance” means a ratio of sound pressure on a surface to sound fluxthrough the surface, the ratio having a reactance and a resistancecomponent. As used herein, “acoustic lens” means a system or device forspreading or converging sounds waves. As used herein, “acousticscattering” means irregular and multi-directional reflection anddiffraction of sound waves produced by multiple reflecting surfaces, thedimensions of which are small compared to the wavelength, or by certaindiscontinuities in the medium through which the wave is propagated. Asused herein, “cavitation” means the nucleation, expansion and decay orcollapse of a vacuum space (cavity) or gas/vapor space (bubble) in afluid as a result of an acoustic pressure field. As used herein,“bubble” means a gas body or cavity in a fluid or at a fluid/solidinterface having in its interior a vacuum, or a gas or mixture ofgasses. As used herein, “couplant” means any single material orplurality of materials in an acoustic path for coupling acoustic energyfrom a source location to another location. A couplant can be a portionof a microdevice, such as a wall of a microdevice, used to coupleacoustic energy from an acoustic source to an internal chamber of themicrodevice. As used herein, “non-contact” refers to an acoustic sourcenot being in mechanical contact with a fluid to be controlled. As usedherein, “active site” means location of a receptor or sensor of anykind, such as, nucleic acid, nucleic acid probe, protein, antibody,small molecule, tissue sample and nonbiological material. As usedherein, “couplant” means a material that conducts acoustic energy from asource to another location.

I. Apparatus and Methods for Ultrasonic Treatment

In certain embodiments, the apparatus includes a source of sonic energy,a sensor for monitoring the energy or its effect, and a feedbackmechanism coupled with the source of sonic energy to regulate the energy(for example, voltage, frequency, pattern) for transmitting ultrasonicenergy to a target. Devices for transmission may include detection andfeedback circuits to control one or more of losses of energy atboundaries and in transit via reflection, dispersion, diffraction,absorption, dephasing and detuning. For example, these devices cancontrol energy according to known loss patterns, such as beam splitting.Sensors can detect the effects of ultrasonic energy on targets, forexample, by measuring electromagnetic emissions, typically in thevisible, IR, and UV ranges, optionally as a function of wavelength.These effects include energy dispersion, scattering, absorption, and/orfluorescence emission. Other measurable variables include electrostaticproperties such as conductivity, impedance, inductance, and/or themagnetic equivalents of these properties. Measurable parameters alsoinclude observation of physical uniformity, pattern analysis, andtemporal progression uniformity across an assembly of treatment vessels,such as a microtiter plate.

As shown in FIG. 1, one or more sensors coupled to a feedback controlresults in more focused, specific, or controlled treatment than thatpossible using current methods typical in the art. The feedbackmethodology can include fixed electronic elements, a processor, acomputer, and/or a program on a computer. The electronic elements,processor, computer, and/or computer program can in turn control any ofa variety of adjustable properties to selectively expose a sample tosonic energy in a given treatment. These properties can includemodulation of the ultrasonic beam in response to a detected effect.Modifiable ultrasonic wave variables can include intensity, duty cycle,pulse pattern, and spatial location. Typical input parameters that cantrigger an output can include change in level of signal, attainment ofcritical level, plateauing of effect, and/or rate of change. Typicaloutput actions can include sonic input to sample, such as frequency,intensity, duty cycle; stopping sample movement or sonic energy; and/ormoving beam within a sample or to the next sample.

More particularly, FIG. 1 depicts an electronically controlledultrasonic processing apparatus 100 that includes an ultrasoundtreatment system and associated electronics 200, a positioning system300 for the sample target 800 being treated, and a control system 400which controls, generates, and modulates the ultrasound signal andcontrols the positioning system 300 in a predetermined manner that mayor may not include a feedback mechanism. The source of sonic energy 230and the target 800 being treated for example, a sample, multiplesamples, or other device are arranged in a fluid bath 600, such aswater, such that the source of sonic energy 230 is oriented towards thetarget 800. The target 800 may be positioned proximate the surface ofthe fluid bath 600, above the source of sonic energy 230, all beingcontained within a sample processing vessel 500. Any of a multitude ofsensors 700 for measuring processing parameters can be arranged in orproximate to the fluid bath 600. A temperature control unit 610 may beused to control the temperature of the fluid in the fluid bath 610. Anoverpressure system 900 can control, for example, cavitation, bymaintaining a positive pressure on the target 800 and may be adjusted,in a predetermined manner that may or may not include feedbackprocessing, by a target pressure controller 910 that is connected to thecontrol system 400.

An ultrasound acoustic field 240 can be generated by the sonic energysource 230, for example, a focused piezoelectric ultrasound transducer,into the fluid bath 600. According to one embodiment, the sonic energysource 230 can be a 70 mm diameter spherically focused transducer havinga focal length of 63 mm, which generates an ellipsoidal focal zoneapproximately 2 mm in diameter and 6 mm in axial length when operated ata frequency of about 1 MHz. The sonic energy source 230 is positioned sothat the focal zone is proximate the surface of the fluid bath 600. Thesonic energy source 230 can be driven by an alternating voltageelectrical signal generated electronically by the control system 400.

The positioning system 300 can include at least one motorized linearstage 330 that allows the target to be positioned according to aCartesian coordinate system. The positioning system 300 may position andmove the target 800 relative to the source 230 in three dimensions (x,y, z) and may optionally move either or both of the target 800 and thesonic energy source 230. The positioning system 300 can move target 800during and as part of the treatment process and between processes, aswhen multiple samples or devices within the target 800 are to beprocessed in an automated or high-throughput format. The positioningsystem 300 may position or move the target 800 in a plane transverse tothe focal axis of the sonic energy source 230 (x- and y-axes). Thepositioning system 300 can position and move the target 800 along thefocal axis of the sonic energy source 230 and lift or lower the target800 from or into the fluid bath 600 (z-axis). The positioning system 300can also position the sonic energy source 230 and any or all of thesensors 700 in the fluid bath 600 along the focal axis of the sonicenergy source 230, if the sensors 700 are not affixed in the water bath600, as well as lift, lower, or otherwise move the sonic energy source230. The positioning system 300 also can be used to move other devicesand equipment such as detection devices and heat exchange devices fromor into the fluid bath 600 (z-axis). The linear stages of thepositioning mechanism 330 can be actuated by stepper motors (not shown),which are driven and controlled by electrical signals generated by thecontrol system 400, or other apparatus known to those skilled in theart.

The control system 400 can include a computer 410 and a userinput/output device or devices 420 such as a keyboard, display, printer,etc. The control system is linked with the ultrasound treatment system200 to drive the sonic energy source 230, with the positioning system300 to drive the stepper motors described above, with one or moresensors 700 to detect and measure process conditions and parameters, andwith one or more controllers, such as the target pressure controller910, to alter conditions to which the target 800 is exposed. A fluidbath controller 610 can also be linked with the control system 400 toregulate temperature of the fluid bath 600. The user interface 420allows an operator to design and specify a process to be performed upona sample. In this regard, the ultrasound treatment system 200 caninclude an arbitrary waveform generator 210 that drives an RF amplifier220, such that the sonic energy source 230 receives an input. The outputsignal of the RF amplifier 220 may be conditioned by an impedancematching network and input to the sonic energy source 230. The computer410 also drives and controls the positioning system 300 through, forexample, a commercially available motion control board 310 and steppermotor power amplifier device 320.

The control system 400 can generate a variety of useful alternatingvoltage waveforms to drive the sonic energy source 230. For instance, ahigh power “treatment” interval consisting of about 5 to 1,000 sinewaves, for example, at 1.1 MHz, may be followed by a low power“convection mixing” interval consisting of about 1,000 to 1,000,000 sinewaves, for example, at the same frequency. “Dead times” or quiescentintervals of about 100 microseconds to 100 milliseconds, for example,may be programmed to occur between the treatment and convection mixingintervals. A combined waveform consisting of concatenated treatmentintervals, convection mixing intervals, and dead time intervals may bedefined by the operator or selected from a stored set of preprogrammedwaveforms. The selected waveform may be repeated a specified number oftimes to achieve the desired treatment result. Measurable or discernibleprocess attributes such as sample temperature, water bath temperature,intensity of acoustic cavitation, or visible evidence of mixing in thesample processing vessel 500, may be monitored by the control system 400and employed in feedback loop to modify automatically the treatmentwaveform during the treatment process. This modification of thetreatment waveform may be a proportional change to one or more of thewaveform parameters or a substitution of one preprogrammed waveform foranother. For instance, if the sample temperature deviates excessivelyduring treatment from a set-point temperature due to absorbed acousticenergy, the control system 400 may proportionally shorten the treatmentinterval and lengthen the convection mixing interval in response to theerror between the actual and target sample temperatures. Or,alternatively, the control system 400 may substitute one predeterminedwaveform for another. The control system 400 may be programmed toterminate a process when one or more of the sensors 700 signal that thedesired process result has been attained.

The control system 400 controls and drives the positioning system 300with the motion control board 310, power amplifier device 320, andmotorized stage 330, such that the target 800 can be positioned or movedduring treatment relative to the source 230 to selectively expose thetarget 800 to sonic energy, described more fully below.

Various aspects of the embodiment of FIG. 1 and of components of theembodiment shown in FIG. 1, as well as other embodiments with the same,similar, and/or different components, are more fully described below.

A. Transducer

In certain embodiments, the sonic energy source 230, for example, anultrasound transducer or other transducer, produces acoustic waves inthe “ultrasonic” frequency range. Ultrasonic waves start at frequenciesabove those that are audible, typically about 20,000 Hz or 20 kHz, andcontinue into the region of megahertz (MHz) waves. The speed of sound inwater is about 1000 meters per second, and hence the wavelength of a1000 Hz wave in water is about a meter, typically too long for specificfocusing on individual areas less than one centimeter in diameter,although usable in non-focused field situations. At 20 kHz thewavelength is about 5 cm, which is effective in relatively smalltreatment vessels. Depending on the sample and vessel volume, preferredfrequencies may be higher, for example, about 100 kHz, about 1 MHz, orabout 10 MHz, with wavelengths, respectively, of approximately 1.0, 0.1,and 0.01 cm. In contrast, for conventional sonication, including sonicwelding, frequencies are typically approximately in the tens of kHz, andfor imaging, frequencies are more typically about 1 MHz and up to about20 MHz. In lithotripsy, repetition rates of pulses are fairly slow,being measured in the hertz range, but the sharpness of the pulsesgenerated give an effective pulse wavelength, or in this case, pulserise time, with frequency content up to about 100 to about 300 MHz, or0.1-0.3 gigahertz (GHz).

The frequency used in certain embodiments of the invention also will beinfluenced by the energy absorption characteristics of the sample or ofthe treatment vessel, for a particular frequency. To the extent that aparticular frequency is better absorbed or preferentially absorbed bythe sample, it may be preferred. The energy can be delivered in the formof short pulses or as a continuous field for a defined length of time.The pulses can be bundled or regularly spaced.

A generally vertically oriented focused ultrasound beam may be generatedin several ways. For example, a single-element piezoelectric transducer,such as those supplied by Sonic Concepts, Woodinville, Wash., that canbe a 1.1 MHz focused single-element transducer, can have a sphericaltransmitting surface that is oriented such that the focal axis isvertical. Another embodiment uses a flat unfocused transducer and anacoustic lens to focus the beam. Still another embodiment uses amulti-element transducer such as an annular array in conjunction withfocusing electronics to create the focused beam. The annular arraypotentially can reduce acoustic sidelobes near the focal point by meansof electronic apodizing, that is by reducing the acoustic energyintensity, either electronically or mechanically, at the periphery ofthe transducer. This result can be achieved mechanically by partiallyblocking the sound around the edges of a transducer or by reducing thepower to the outside elements of a multi-element transducer. Thisreduces sidelobes near the energy focus, and can be useful to reduceheating of the vessel. Alternatively, an array of small transducers canbe synchronized to create a converging beam. Still another embodimentcombines an unfocused transducer with a focusing acoustic mirror tocreate the focused beam. This embodiment can be advantageous at lowerfrequencies when the wavelengths are large relative to the size of thetransducer. The axis of the transducer of this embodiment can behorizontal and a shaped acoustic mirror used to reflect the acousticenergy vertically and focus the energy into a converging beam.

In certain embodiments, the focal zone can be small relative to thedimensions of the treatment vessel to avoid heating of the treatmentvessel. In one embodiment, the focal zone has a radius of approximately1 mm and the treatment vessel has a radius of at least about 5 mm.Heating of the treatment vessel can be reduced by minimizing acousticsidelobes near the focal zone. Sidelobes are regions of high acousticintensity around the focal point formed by constructive interference ofconsecutive wavefronts. The sidelobes can be reduced by apodizing thetransducer either electronically, by operating the outer elements of amulti-element transducer at a lower power, or mechanically, by partiallyblocking the acoustic waves around the periphery of a single elementtransducer. Sidelobes may also be reduced by using short bursts, forexample in the range of about 3 to about 5 cycles in the treatmentprotocol.

The transducer can be formed of a piezoelectric material, such as apiezoelectric ceramic. The ceramic may be fabricated as a “dome”, whichtends to focus the energy. One application of such materials is in soundreproduction; however, as used herein, the frequency is generally muchhigher and the piezoelectric material would be typically overdriven,that is driven by a voltage beyond the linear region of mechanicalresponse to voltage change, to sharpen the pulses. Typically, thesedomes have a longer focal length than that found in lithotripticsystems, for example, about 20 cm versus about 10 cm focal length.Ceramic domes can be damped to prevent ringing. The response is linearif not overdriven. The high-energy focus of one of these domes istypically cigar-shaped. At 1 MHz, the focal zone is about 6 cm long andabout 2 cm in diameter for a 20 cm dome, or about 15 mm long and about 3mm wide for a 10 cm dome. The peak positive pressure obtained from suchsystems is about 1 MPa (mega Pascal) to about 10 MPa pressure, or about150 PSI (pounds per square inch) to about 1500 PSI, depending on thedriving voltage.

The wavelength, or characteristic rise time multiplied by sound velocityfor a shock wave, is in the same general size range as a cell, forexample about 10 to about 40 micron. This effective wavelength can bevaried by selection of the pulse time and amplitude, by the degree offocusing maintained through the interfaces between the source and thematerial to be treated, and the like.

In certain embodiments, the focused ultrasound beam is orientedvertically in a water tank so that the sample may be placed at or nearthe free surface. The ultrasound beam creates shock waves at the focalpoint. In an embodiment to treat industry standard microplates whichhold a plurality of samples in an array, a focal zone, defined as havingan acoustic intensity within about 6 dB of the peak acoustic intensity,is formed around the geometric focal point. This focal zone has adiameter of approximately 2 mm and an axial length of about 6 mm.

Ceramic domes are adaptable for in vitro applications because of theirsmall size. Also, systems utilizing ceramic domes can be produced atreasonable cost. They also facilitate scanning the sonic beam focus overa volume of liquid, by using microactuators which move a retainingplatform to which the sample treatment vessel is attached.

Another source of focused pressure waves is an electromagnetictransducer and a parabolic concentrator, as is used in lithotripsy. Theexcitation tends to be more energetic, with similar or larger focalregions. Strong focal peak negative pressures of about −16 MPa have beenobserved. Peak negative pressures of this magnitude provide a source ofcavitation bubbles in water, which can be desirable in an extractionprocess.

The examples described below use a commercial ultrasonic driver using apiezoelectric ceramic, which is stimulated by application of fluctuatingvoltages across its thickness to vibrate and so to produce acousticwaves. These may be of any of a range of frequencies, depending on thesize and composition of the driver. Such drivers are used inlithotripsy, for example, as well as in acoustic speakers and inultrasound diagnostic equipment, although without the control systems asdescribed herein.

These commercially-available drivers have a single focus. Therefore, totreat, for example, to stir, an entire microplate with such a device, itis typically necessary to sequentially position or step each well at thefocus of the driver. Because stirring time is brief, the stepping of a96 well plate can be accomplished in approximately two minutes or lesswith simple automatic controls, as described below. It is contemplatedthat this time can be shortened.

It also is possible to make multi-focal drivers by making piezoelectricdevices with more complex shapes. Modulators of the acoustic fieldattached to an existing piezoelectric driver can also produce multiplefoci. These devices can be important for obtaining rapid throughput ofmicroplates in a high density format, such as the 1534-well format.

B. Drive Electronics and Waveform Control.

One treatment protocol can include variable acoustic waveforms combinedwith sample motion and positioning to achieve a desired effect. Theacoustic waveform of the transducer has many effects, including:acoustic microstreaming in and near cells due to cavitation, that isflow induced by, for example, collapse of cavitation bubbles; shockwaves due to nonlinear characteristics of the fluid bath; shock wavesdue to cavitation bubbles; thermal effects, which lead to heating of thesample, heating of the sample vessel, and/or convective heat transferdue to acoustic streaming; flow effects, causing deflection of samplematerial from the focal zone due to shear and acoustic pressure, as wellas mixing due to acoustic streaming, that is flow induced by acousticpressure; and chemical effects.

The treatment protocol can be optimized to maximize energy transferwhile minimizing thermal effects. The treatment protocol also caneffectively mix the contents of the treatment vessel, in the case of aparticulate sample suspended in a liquid. Energy transfer into thesample can be controlled by adjusting the parameters of the acousticwave such as frequency, amplitude, and cycles per burst. Temperaturerise in the sample can be controlled by limiting the duty cycle of thetreatment and by optimizing heat transfer between the treatment vesseland the water bath. Heat transfer can be enhanced by making thetreatment vessel with thin walls, of a relatively highly thermallyconductive material, and/or by promoting forced convection by acousticstreaming in the treatment vessel and in the fluid bath in the proximityof the treatment vessel. Monitoring and control of temperature isdiscussed in more detail below.

For example, for a cellular disruption and extraction treatment, anexample of an effective energy waveform is a high amplitude sine wave ofabout 1000 cycles followed by a dead time of about 9000 cycles, which isabout a 10% duty cycle, at a frequency of about 1.1 MHz. The sine waveelectrical input to the transducer typically results in a sine waveacoustic output from the transducer. As the focused sine waves convergeat the focal point, they can become a series of shock waves due to thenonlinear acoustic properties of the water or other fluid in the bath.This protocol treats the material in the focal zone effectively duringthe “on” time. As the material is treated, it typically is expelled fromthe focal zone by acoustic shear and streaming. New material circulatesinto the focal zone during the “off” time. This protocol can beeffective, for example, for extracting the cellular contents of groundor particulate leaf tissue, while causing minimal temperature rise inthe treatment vessel.

Further advantage in disruption and other processes may be gained bycreating a high power “treat” interval 10 alternating with a low power“mix” interval 14, as shown schematically in FIG. 2. More particularly,in this example, the “treat” interval 10 utilizes a sine wave that has atreatment frequency 18, a treatment cycles-per-burst count 26, and atreatment peak-to-peak amplitude 22. The “mix” interval 14 has a mixfrequency 20, a mix cycles-per-burst count 28 and a lower mixpeak-to-peak amplitude 24. Following each of the intervals 10, 14 is adead time 12, 16. Of course, these relationships are merely one exampleof many, where one interval in considered to be high power and oneinterval is considered to be low power, and these variables and otherscan be altered to produce more or less energetic situations.Additionally, the treat function or interval and the mix function orinterval could emit from different or multiple transducers in the sameapparatus, optionally emitting at different frequencies.

High power/low power interval treatments can allow multiple operationsto be performed, such as altering permeability of components, such ascells, within the sample followed by subsequent mixing of the sample.The treat interval can maximize cavitation and bioeffects, while the mixinterval can maximize mixing within the treatment vessel and/or generateminimal heat. Adding a longer, high power “super-mix” intervaloccasionally to stir up particles that are trapped around the peripheryof the treatment vessel can provide further benefits. This “super-mix”interval generates additional heat, so it is programmed to treatinfrequently during the process, for example, every few seconds.Additionally, dead times between the mix and treat intervals, duringwhich time substantially no energy is emitted from the sonic energysource, can allow fresh material to circulate into the energy focal zoneof the target.

As discussed below, moving the sample vessel during treatment relativeto the source, so that the focal zone moves within the treatment vessel,can further enhance the process. For example, target motion through thefocal zone can resuspend material in the sample that may have clumped orbecome trapped around the periphery of the treatment vessel. A similarimprovement can be achieved by traversing or “dithering” the treatmentvessel relative to the focal zone, described more fully below withrespect to FIG. 3. Dithering can become increasingly advantageous as thesample treatment vessel becomes significantly larger than the focalzone.

The waveform of focused sound waves can be a single shock wave pulse, aseries of individual shock wave pulses, a series of shock wave bursts ofseveral cycles each, or a continuous waveform. Incident waveforms can befocused directly by either a single element, such as a focused ceramicpiezoelectric ultrasonic transducer, or by an array of elements withtheir paths converging to a focus. Alternatively, multiple foci can beproduced to provide ultrasonic treatment to multiple treatment zones,vessels, or wells.

Reflected waveforms can be focused with a parabolic reflector, such asis used in an “electromagnetic” or spark-gap type shock-wave generator.Incident and reflected waveforms can be directed and focused with anellipsoidal reflector such as is used in an electrohydraulic generator.Waveforms also can be channeled.

The waveform of the sound wave typically is selected for the particularmaterial being treated. For example, to enhance cavitation, it can bedesirable to increase the peak negative pressure following the peakpositive pressure. For other applications, it can be desirable to reducecavitation but maintain the peak positive pressure. This result can beachieved by performing the process in a pressurized chamber at a slightpressure above ambient. For example, if the waveform generated has apeak negative pressure of about −5 MPa, then the entire chamber may bepressurized to about 10 MPa to eliminate cavitation from occurringduring the process. Liquid to be treated can be pressurized on a batchor a continuous basis.

A variety of methods of generating waves can be used. In lithotripsy,for example, “sharp” shock waves of high intensity and short durationare generated. Shock waves may be generated by any method that isapplicable to a small scale. Such methods include spark dischargesacross a known gap; laser pulses impinging on an absorptive orreflective surface; piezoelectric pulses; electromagnetic shock waves;electrohydraulic shock waves created by electrical discharges in aliquid medium; and chemical explosives. In the case of explosives,microexplosives in wells in a semiconductor-type chip can be fabricatedin which the wells are individually addressable. Also, amagnetostrictive material can be exposed to a magnetic field, and it canexpand and/or contract such that the material expansion/contractioncreates sonic energy.

Continuous sinusoidal sound waves can be generated by any process thatis appropriate for focusing on a small scale. For example, ceramicpiezoelectric elements may be constructed into dome shapes to focus thesound wave into a point source. In addition, two or more shock waves maybe combined from the same source, such as piezoelectric elementsarranged in mosaic form, or from different sources, such as anelectromagnetic source used in combination with a piezoelectric source,to provide a focused shock wave.

Typically, the shock wave is characterized by a rapid shock front with apositive peak pressure in the range of about 15 MPa, and a negative peakpressure in the range of about negative 5 MPa. This waveform is of abouta few microseconds duration, such as about 5 microseconds. If thenegative peak is greater than about 1 MPa, cavitation bubbles may form.Cavitation bubble formation also is dependent upon the surroundingmedium. For example, glycerol is a cavitation inhibitive medium, whereasliquid water is a cavitation promotive medium. The collapse ofcavitation bubbles forms “microjets” and turbulence that impinge on thesurrounding material.

The waves are applied to the samples either directly, as for example,piezoelectric pulses, or via an intervening medium. This medium can bewater or other fluid. An intervening medium also can be a solid, such asa material which is intrinsically solid or a frozen solution. Waves alsocan be applied through a container, such as a bottle, bag, box, jar, orvial.

For maximum control, and particularly for well-by-well mixing, a focusedacoustic pulse is useful. When a pulse is emitted from a curved sourcewith an elliptical profile, then the emitted acoustic waves or pulsesfocus in a small region of maximum intensity. The location of the focuscan be calculated or determined readily by experiment. The diameter ofthe focal zone can be of the same general size as or smaller than thediameter of the treatment vessel. Then, mixing energy can be provided toeach well for a repeatable amount of time, providing uniform mixing ofeach sample.

C. X-Y-Z Cartesian Positioning System.

In certain embodiments, the sample is not only moved into positionrelative to the transducer initially, but positioned during treatment toinsure uniform treatment of the sample, where the sample is kept wellsuspended during treatment. As used herein, x and y axes define a planethat is substantially horizontal relative to ground and/or a base of anapparatus of the invention, while the z axis lies in a plane that issubstantially vertical relative to the ground and/or the base of anapparatus and perpendicular to the x-y plane.

One positioning scheme is termed “dithering,” which entails slightlyvarying the position of the sample relative to the source which canoccur by moving the sample through the focal zone in several ways. Forexample, but without limitation, the sample can be moved in a circle, oroval, or other arcuate path with a certain radius 30 and moved a certaindistance 34 in certain increments or steps 32, as depicted schematicallyin FIG. 3. These movements can vary between treatment cycles or during aparticular treatment cycle and have several effects. First, ditheringthe sample position sweeps the focal zone through the volume of thesample treatment vessel or device, treating material that is notinitially in the focal zone. In addition, varying the location of theacoustic focus within the vessel tends to make treatment, and theresulting heating, more uniform within each sample.

Certain embodiments include drive electronics and devices forpositioning of the sample(s). In one embodiment, the positioningsequence, optionally including dithering, and the treatment pulse trainare pre-programmed, for example in a computer, and are executedautomatically. The driver electronics and positioners can be linkedthrough the control system to sensors so that there is “real time”feedback of sensor data to the control system during treatment in orderto adjust the device(s) for positioning the sample and prevent localizedheating or cavitation. The drive electronics can include a waveformgenerator matching network, an RF switch or relay, and a radio frequency(RF) amplifier, for safety shutdown.

The positioning system can include a three axis Cartesian positioningand motion control system to position the sample treatment vessel or anarray of sample treatment vessels relative to the ultrasound transducer.The “x” and “y” axes of the Cartesian positioning system allow eachsample in an array of samples, such as an industry standard microplate,to be brought into the focal zone for treatment. Alternativeconfigurations may employ a combination of linear and rotary motioncontrol elements to achieve the same capabilities as the three axisCartesian system. Alternative positioning systems may be constructed ofself-contained motor-driven linear or rotary motion elements mounted toeach other and to a base plate to achieve two- or three-dimensionalmotion.

As used in the examples, stepper motors, such as those available fromEastern Air Devices, located in Dover, N.H., drive linear motionelements through lead screws to position the sample. The stepper motorsare driven and controlled by means of LabVIEW software controlling aValueMotion stepper motor control board available from NationalInstruments, located in Austin, Tex. The output signals from the controlboard are amplified by a nuDrive multi-axis power amplifier interface,also available from National Instruments, to drive the stepper motors.

The computer controlled positioning system can be programmed tosequentially move any defined array of multiple samples into alignmentwith the focal zone of the ultrasound transducer. If temperature riseduring treatment is an issue, the samples in a multi-sample array can bepartially treated and allowed to cool as the positioning systemprocesses the other samples. This can be repeated until all the sampleshave been treated fully.

The positioning system also can move the sample treatment vesselrelative to the focal point during treatment to enhance the treatment orto treat a sample that is large relative to the focal zone. By sweepingthe sample slowly in a circular or other motion during treatment, clumpsof material around the periphery of the treatment vessel may be brokenup advantageously. In addition, x-y dithering may prevent a “bubbleshield” from forming and blocking cavitation in the sample treatmentvessel. The x-y dithering may also enhance treatment of samplesuspensions that have a high viscosity or become more viscous duringtreatment and do not mix well. The sample position may also be ditheredvertically in the z-axis. This may be advantageous in a deep treatmentvessel where the depth is substantially larger than the axial dimensionof the focal zone, in order to treat the entire contents of thetreatment vessel or to resuspend larger sample fragments which have sunkto the bottom of the vessel. Dithering in all three dimensions may alsobe employed, as depicted in FIG. 3

For a relatively flat sample, such as whole leaf tissue, a histologicalsample, or thin-section specimen, where the area of the sample is largerelative to the cross-sectional area of the focal zone, the x-ypositioning system can cause the focal zone to traverse the sample inorder to treat the entire surface of the sample. This procedure may becombined with optical analysis or other sensors to determine the extentof the treatment to each portion of the sample that is brought into thefocal zone.

In certain embodiments, the sample or array of samples can be movedrelative to the transducer and the other parts of the apparatus. Inalternative embodiments the transducer is moved while the sample holderremains fixed, relative to the other parts of the apparatus. As analternative, movement along two of the axes, for example, x and y, canbe assigned to the sample holder and movement along the third axis, z inthis case, can be assigned to the transducer.

The three axis positioning system enables automated energy focusadjustment in the z-axis when used in conjunction with a sensor formeasuring the ultrasound intensity. In one embodiment, a needlehydrophone can be mounted in a fixture on the sample positioning system.The hydrophone can be traversed in three dimensions through the focalregion to record the acoustic intensity as a function of position inorder to map out the focal zone. In another embodiment, a number ofpositions on a sheet of aluminum foil held in the sample holder can betreated in a sequence of z-axis settings. The foil can then be examinedto determine the spot size of the damage at each position. The diameterof the spot corresponds generally to the diameter of the focal zone atthat z-axis setting. Other, fully automated embodiments of a focusingsystem can also be constructed.

The three axis positioning system also allows the apparatus to beintegrated into a larger laboratory automation scheme. A positioningsystem with an extended work envelope can transfer microplates or othersample vessels into and out of the apparatus. This allows the apparatusto interact automatically with upstream and downstream processes.

D. Sensors

Visual Monitoring of the Sample

Optical or video detection and analysis can be employed to optimizetreatment of the sample. For example, in a suspension of biologicaltissue, the viscosity of the mixture can increase during treatment dueto the diminution of the particles by the treatment and/or by theliberation of macromolecules into the solution. Video analysis of thesample during treatment allows an automated assessment of the mixingcaused by the treatment protocol. The protocol may be modified duringthe treatment to promote greater mixing as a result of this assessment.The video data may be acquired and analyzed by the computer controlsystem that is controlling the treatment process. Other opticalmeasurements such as spectral excitation, absorption, fluorescence,emission, and spectral analysis also can be used to monitor treatment ofthe sample. A laser beam, for example, can be used for alignment and toindicate current sample position.

Monitoring of Temperature

Heating of individual wells can be determined by an infraredtemperature-sensing probe, collimated so as to view only the well beingtreated with the ultrasonic energy. For example, an infrared thermalmeasuring device can be directed at the top unwetted side of thetreatment vessel. This provides a non-contact means of analysis that isnot readily achievable in conventional ultrasound treatmentconfigurations. The thermal information can be recorded as a thermalrecord of the sample temperature profile during treatment.

Active temperature monitoring may be used as a feedback mechanism tomodify the treatment protocol during the treatment process to keep thesample temperature within specified limits. For example, an infraredsensor directed at the sample treatment vessel may input temperaturereadings to the computer. The computer, in accordance with a controllingprogram, can produce output directed to the circuit enabling theultrasonic transducer, which in turn can reduce the high power treatmentintervals and increase the low power mixing intervals, for example, ifthe sample temperature is nearing a specified maximum temperature.

Monitoring of Cavitation

A variety of methods may be employed to detect cavitation. For example,acoustic emissions, optical scattering, high-speed photography,mechanical damage, and sonochemicals can be used. As described above formonitoring temperature, information from cavitation detection can beused by the system to produce an output that selectively controlsexposure of a sample to sonic energy in response to the information.Each of these methods to monitor cavitation are described more fullybelow.

Acoustic emissions: Bubbles are effective scatterers of ultrasound. Thepulsation mode of a bubble is referred to as monopole source, which isan effective acoustic source. For small, generally linear oscillations,the bubble simply scatters the incident acoustic pulse. However, as theresponse becomes more nonlinear, it also starts to emit signals athigher harmonics. When driven harder, the bubbles start to generatesubharmonics as well. Eventually as the response becomes a periodic orchaotic, the scattered field tends towards white noise. In the scenariowhere inertial collapses occur, short acoustic pressure pulses areemitted. An acoustic transducer can be configured to detect theseemissions. There is a detectable correlation between the onset of theemissions and cell disruption.

Optical scattering: Bubbles also scatter light. When bubbles arepresent, light is scattered. Light can normally be introduced into thesystem using fiber optic light sources so that cavitation can bedetected in real-time, and therefore can be controlled by electronic andcomputer systems.

High-speed photography: Bubbles can be photographed. This methodtypically requires high-speed cameras and high intensity lighting,because the bubbles respond on the time frame of the acoustics. It alsorequires good optical access to the sample under study. This method cangive detailed and accurate data and may be a consideration whendesigning systems according to the invention. Stroboscopic systems,which take images far less frequently, can often give similarqualitative performance more cheaply and easily than high-speedphotography.

Mechanical damage: Cavitation is known to create damage to mechanicalsystems. Pitting of metal foils is a particularly common effect, anddetection method. There is a correlation between the cavitation neededto pit foils and to disrupt cells.

Sonochemicals: A number of chemicals are known to be produced inresponse to cavitation. The yield of these chemicals can be used as ameasure of cavitational activity. A common technique is to monitor lightgeneration from chemicals, such as luminol, that generate light whenexposed to cavitation. Sonochemical yield usually can not be done duringcell experiments but can be done independently under identicalconditions, and thereby, provide a calibrated standard.

E. Temperature, Cavitation, and Pressure Management and Control.

Temperature Control

Certain applications require that the temperature of the sample beingprocessed be managed and controlled during processing. For example, manybiological samples should not be heated above 4° C. during treatment.Other applications require that the samples be maintained at a certainelevated temperature during treatment. The ultrasound treatment protocolinfluences the sample temperature in several ways: the sample absorbsacoustic energy and converts it to heat; the sample treatment vesselabsorbs acoustic energy and converts it to heat which, in turn, can heatthe sample; and acoustic streaming develops within the sample treatmentvessel and the water bath, forcing convective heat transfer between thesample treatment vessel and the water bath. In the case of a relativelycool water bath, this cools the sample.

The acoustic waves or pulses can be used to regulate the temperature ofthe solutions in the treatment vessel. At low power, the acoustic energyproduces a slow stirring without marked heating. Although energy isabsorbed to induce the stirring, heat is lost rapidly through the sidesof the treatment vessel, resulting in a negligible equilibriumtemperature increase in the sample. At higher energies, more energy isabsorbed, and the temperature rises. The degree of rise per unit energyinput can be influenced and/or controlled by several characteristics,including the degree of heat absorption by the sample or the treatmentvessel and the rate of heat transfer from the treatment vessel to thesurroundings. Additionally, the treatment protocol may alternate ahigh-powered treatment interval, in which the desired effects areobtained, with a low power mixing interval, in which acoustic streamingand convection are achieved without significant heat generation. Thisconvection may be used to promote efficient heat exchange or cooling.

The thermal information can also be used to modify or control thetreatment to maintain the sample temperature rise below a maximumallowable value. The treatment can be interrupted to allow the sample tocool down. In certain embodiments, the output of the thermal measurementdevice or system is entered into the computer control system forrecording, display on a control console, and/or control of exposure ofthe sample to sonic energy through a feedback loop, for example byaltering the duty cycle.

Temperature rise during ultrasonic continuous wave exposure can becontrolled, if required, by refrigeration of a liquid or other samplebefore, during, or after passage through a zone of sonic energy, ifprocessing in a continuous, flow-through mode. In generally stationarydiscrete sample processing modes, a sample can be cooled by air, bycontact with a liquid bath, or a combination of both air and liquid. Thetemperature is rapidly equilibrated within the vessel by the stirringaction induced by the acoustic waves. As a result, and especially insmall vessels or other small fluid samples, the rate of temperatureincrease and subsequent cooling can be very rapid. The rate of deliveryof sonic energy to the material can also be controlled, although thatcan lengthen processing time.

Liquids within the sample can be provided at any temperature compatiblewith the process. The liquid may be frozen or partially frozen forprocessing. For example, when biological material is subjected tosubzero temperatures below about −5° C., most, but not all, of the wateris in the solid phase. However, in certain biological tissues,micro-domains of liquid water still remain for several reasons, such asnatural “antifreeze” molecules or regions of higher salt concentration.Therefore, sample temperature may be varied during the procedure. Atemperature is selected at which microdomains of liquid water are ableto form shock wave induced cavitation due to bubble formation andcollapse, resulting in shear stresses that impinge on surroundingtissues. Indeed, gradually altering the sample temperature can bedesirable, as it provides focused domains of liquid water for collectionof sonic energy for impingement on the surrounding material.

Treatment baths can be relatively simple, and can include a water bathor other fluid bath that is employed to conduct the acoustic waves fromthe transducer to the sample treatment vessel, where the liquid istemperature controlled. In certain embodiments, the entire bath ismaintained at a specific temperature by means of an external heater orchiller, such as a Neslab RTE-210 chiller available from NeslabInstruments, Inc., located in Newington, N.H., and heat exchanger coilsimmersed in the bath. The sides and bottom of the tank containing thebath may have sufficient insulating properties to allow the bath to bemaintained substantially uniformly at a specific temperature. Anotherembodiment, such as that depicted in FIG. 7, employs an inner tray orsample tank 76 made of an insulating material such as rigid polystyrenefoam which is set within a larger water bath 84 in a transducer tank 82.The inner tray 76 has heat-exchanger tubes or other heating or coolingdevices within it (not shown) to allow a fluid 78 such as ethyleneglycol or propylene glycol in the inner tray 76 to be heated or cooledbeyond what may be practical for the fluid 84 such as water in the outerbath in the transducer tank 82. The inner tray 76 has an acoustic window88 in the bottom. The acoustic window 88 is made of a thin film materialhaving low acoustic absorption and an acoustic impedance similar towater. This inner tray 76 is arranged so that the acoustic window 88 isaligned with a transducer 86 which is outside the tray 76, supportedwith a support 80 in the water 84. A sample 74 is located within amicrotiter plate or other sample treatment vessel 72, within the tray 76and is subjected to the thermal influence of the inner treatment bath78. The treatment vessel 70 can be movable relative to the transducer 86with a positioning system 70. Also, sonic energy focuses on the sample74 through the acoustic window 88. This arrangement permits the use ofseparate fluids and substantially independent control of the temperatureof the inner 76 and outer treatment baths 84. The smaller volume of theinner tray 76 facilitates the use of antifreeze mixtures, such as amixture of propylene glycol and water, at temperatures below thefreezing temperature of water. This, in turn, allows the samples 74 tobe processed and treated at temperatures below the freezing temperatureof water. This embodiment is beneficial for treatment applicationsrequiring that the sample materials 74 be maintained at temperaturesnear or below the freezing point of water. It allows for the containmentof treatment bath fluids 78, such as antifreeze solutions, that may notbe compatible with the transducer 86 and other system components. Italso allows the transducer 86 to be maintained at a differenttemperature than the samples 74. This embodiment may also be connectedwith any of the other components described in FIG. 1 and is suitable foruse in a system with or without feedback loop control.

Sample temperature may be required to remain within a given temperaturerange during a treatment procedure. Temperature can be monitoredremotely by, for example, an infra-red sensor. Temperature probes suchas thermocouples may not be particularly well suited for allapplications because the sound beam may interact with the thermocoupleand generate an artificially high temperature in the vicinity of theprobe. Temperature can be monitored by the same computer that controlsacoustic waveform. The control responds to an error signal which is thedifference between the measured actual temperature of the sample and thetarget temperature of the sample. The control algorithm can be as ahysteritic bang-bang controller, such as those in kitchen stoves, where,as an output of the control system, the acoustic energy is turned offwhen the actual temperature exceeds a first target temperature andturned on when the actual temperature falls below a second targettemperature that is lower than the first target temperature. Morecomplicated controllers can be implemented. For example, rather thansimply turning the acoustic signal on and off, the acoustic signal couldcontinuously be modulated proportionally to the error signal, forexample, by varying the amplitude or the duty cycle, to provide finertemperature regulation.

In the application of a bang-bang control algorithm for a multiplesample format, once a maximum temperature value has been exceeded andthe sonic energy is turned off for a particular sample, an alternativeto waiting for the sample to cool below a selected temperature beforeturning the sonic energy on again, is to move on to the next sample.More particularly, some of the samples can be at least partially treatedwith sonic energy, in a sequence, and then, the system can return to thepreviously partially treated samples to take a sensor reading todetermine if the samples have cooled below the selected temperature andto reinitiate treatment if they have. This procedure treats the samplesin an efficient manner and reduces the total treatment time for treatingmultiple samples. Another alternative is to switch to a predefined“cooling” waveform which promotes convection without adding significantheat to a particular sample, rather than moving on to the next sampleand returning to the first sample at a later time.

If uniformity of temperature throughout the sample is important, thencontrol techniques can be used to ensure a uniform temperaturedistribution. An array of infra-red sensors can be used to determine thedistribution of the temperature inside the sample. If areas of increasedtemperature relative to the rest of the sample appear, then thetransducer can be switched from high power “treatment” mode to low power“mixing” mode. In the low power “mixing” mode, the sample isacoustically stirred until the sample is substantially uniform intemperature. Once temperature uniformity is achieved, the high power“treatment” mode is reinitiated. A control system can monitortemperature and responsively turn the various modes on or off. Whencontrolled by a computer, the intervals during which these modes areused can be very short, for example fractions of a second, thereby notsignificantly prolonging treatment times. Stepping times between wells,or other sample containers, can also be less than a second with suitabledesign.

Cavitation Control

In some applications, it can be preferable to treat the sample with asmuch energy as possible without causing cavitation. This result can beachieved by suppressing cavitation. Cavitation can be suppressed bypressurizing the treatment vessel above ambient, often known as“overpressure,” to the point at which no negative pressure developsduring the rarefaction phase of the acoustic wave. This suppression ofcavitation is beneficial in applications such as cell transformationwhere the desired effect is to open cellular membranes while maintainingviable cells. In other applications it may be desirable to enhancecavitation. In these applications, a “negative” overpressure or vacuumcan be applied to the region of the focal zone.

The control of cavitation in the sample also can be important duringacoustic treatment processes. In some scenarios, the presence of smallamounts of cavitation may be desirable to enhance biochemical processes;however, when large numbers of cavitation bubbles exist they can scattersound before it reaches the target, effectively shielding the sample.

Cavitation can be detected by a variety of methods, including acousticand optical methods. An example of acoustic detection is a passivecavitation detector (PCD) which includes an external transducer thatdetects acoustic emissions from cavitation bubbles. The signal from thePCD can be filtered, for example using a peak detector followed by a lowpass filter, and then input to a controlling computer as a measure ofcavitation activity. The acoustic signal could be adjusted in wayssimilar to those described in the temperature control example tomaintain cavitation activity at a desired level.

Overpressure: Increased ambient pressure is one technique forcontrolling cavitation. Overpressure tends to remove cavitation nuclei.Motes in the fluid are strongly affected by overpressure and socavitation in free-fluid is often dramatically reduced, even by theaddition of one atmosphere of overpressure. Nucleation sites oncontainer walls tend to be more resistant to overpressure; however thecavitation tends to be restricted to these sites and any gas bubblesthat float free into the free-fluid are quickly dissolved. Thereforecells in the bulk fluid are typically unaffected by cavitation sitesrestricted to the container walls. Overpressure may be applied to thetreatment vessel, the array of treatment vessels, the treatment bath andtank, or to the entire apparatus to achieve a higher than atmosphericpressure in the region of the focal zone.

Degassing: Reducing the gas content of the fluid tends to reducecavitation, again by reducing cavitation nuclei and making it harder toinitiate cavitation. Another method of controlling cavitation or theeffects of cavitation is to control the gasses that are dissolved in thesample fluid. For instance, cavitation causes less mechanical damage influid saturated with helium gas than in fluid saturated with argon gas.

Filtering: Cleaner fluids tend to be harder to cavitate.

Various fluids: Certain fluids are much harder to cavitate. Castor oiland mineral oil are nearly cavitation free. Two possible reasons arethat the fluids are of a nature that they tend to fill in cracks, andthat their viscosity also makes them more resistant to cavitation. Thefluids, however, are not particularly compatible with cell preparations.

Waveform shape: The cavitation field responds to the acoustic drivingpulse. It is possible to control the cavitation response, to someextent, by controlling the driving acoustic pressure. Cavitation mayalso be reduced or eliminated by reducing the number of cycles in eachburst of acoustic energy. The cavitation bubbles grow over severalcycles then collapse creating cavitation effects. By limiting the numberof cycles in each burst, bubble growth and collapse can be substantiallyavoided.

F. Treatment or Reaction Vessel

Treatment vessels are sized and shaped as appropriate for the materialto be treated. They can be any of a variety of shapes. For example, asshown in FIGS. 4A-4C, treatment vessels 502, 504, 506 can have verticalwalls, can have a conical shape, or can have a curved shape,respectively. As shown in FIGS. 5A-5C, certain treatment vessel 502,506, prior to treatment with sonic energy, have an upper member 530 anda lower member 550 which together form an interior region that containsthe material 540 to be treated. In certain embodiments, the ultrasoundtransducer projects a focused ultrasound beam upwards. The ultrasoundbeam penetrates the lower member 550 of the treatment vessel 502, 506 toact upon the contents 540 of the treatment vessel 502, 506. The uppermember 530 serves to contain the contents 540 of the vessel 502, 506.

The lower member 550 of the treatment vessel 502, 506 is configured totransmit the maximum amount of ultrasound energy to the contents 540 ofthe vessel 502, 506, minimize the absorption of ultrasound energy withinthe walls of the vessel 502, 506 and maximize heat transfer between thecontents 540 of the treatment vessel 502, 506 and, for example, anexternal water bath. In certain embodiment of the pretreatment assembly,the treatment vessel is thermoformed from a thin film in a hemisphericalshape. The film should have an acoustic impedance similar to that ofwater and low acoustic absorption. One preferred material is low densitypolyethylene. Alternative materials include polypropylene, polystyrene,poly(ethylene teraphthalate) (“PET”), and other rigid and flexiblepolymers. The film may be a laminate to facilitate thermal bonding, forexample using heat sealing. Thicker, more rigid materials may also beemployed. Available multi-well plates in industry standard formats suchas 96 well and 24 well formats may be employed with or withoutmodification. Industry standard thick-wall, multi-well plates with thinfilm bottoms may also be employed. These can work particularlyadvantageously where the size of the focal zone of the ultrasound beamis smaller than a well. In this case, little energy is absorbed by thesides of the treatment vessel and, as a result, relatively little energyis converted to heat.

The upper member of the treatment vessel contains the contents in thevessel during treatment and can act also as an environmental seal. Theupper member of the treatment vessel can be flat or domed to enclose theinterior of the treatment vessel. The upper member of the treatmentvessel may be made of a rigid or flexible material. Preferably, thematerial will have low acoustic absorption and good heat transferproperties. In certain embodiments of the pre-treatment assembly, theupper member of the treatment vessel is a thin film that can be bondedto the lower member, and the lower or upper member can be easilyrupturable for post-treatment transfer of the treated material.

The upper and lower members of the treatment vessel may be joinedtogether by thermal bonding, adhesive bonding, or external clamping.Such joining of the upper and lower members can serve to seal thecontents of the vessel from contaminants in the external environmentand, in an array of vessels, prevent cross-contamination betweenvessels. If the bond is to be achieved by thermal bonding, the upper andlower members of the treatment vessels may be made of film laminateshaving heat bondable outer layers and heat resistant inner layers.

The treatment vessel may be configured as a single unit, as amultiplicity of vessels in an array, or as a single unit with variouscompartments. The upper and lower members of the vessel or array ofvessels can be used once or repeatedly. There also can be a separateframe or structure (not shown) that supports and/or stiffens the upperand lower members of the vessel(s).

This frame or structure may be integral with the vessels or may be aseparate member. An array of treatment vessels may be configured tomatch industry standard multi-well plates. In one embodiment, thetreatment vessel is configured in an array that matches standard 96 wellor 24 well multi-well plates. The frame or supporting structure holdingthe array of treatment vessels can have the same configuration anddimensions as standard multi-well plates.

As shown in FIGS. 6A and 6B, a treatment vessel 508 can include a funnel592 to facilitate transfer of the contents 540 from the treatment vessel508 to a separate vessel 598 after treatment. The funnel 592 can have aconical shape and include an opening at the narrow end. The funnel 592can be rigid, relative to the upper 530 and lower members 550 of thetreatment vessel 508. The large end of the funnel 592 is proximate theupper member 550 of the treatment vessel 508 and aligned with thetreatment vessel 508. The volume of the funnel 592 can be marginallyless than the volume of the treatment vessel 508.

One process of transferring the contents 540 of the treatment vessel 508to another post-treatment vessel 598 includes the following steps. Theupper member 530 of the treatment vessel 508 may be pierced with a sharpinstrument or ruptured when a vacuum is applied. To facilitate rupture,the member 530 may be manufactured from a thin fragile material or madeweak by etching a feature into the surface. Then, the treatment vessel508 is inverted over the post-treatment vessel 598 in a vacuum fixture.A filter 594 may be placed between the treatment vessel 508 and thepost-treatment vessel 598 to separate solids 596 from the liquid 542that is removed from the treatment vessel 508. Alternatively, the filter594 may be incorporated into the outlet of the funnel 592. Thisarrangement of treatment vessel 508 and funnel 592 may be configured asa single unit or as an array of units. This array may match an industrystandard. The treatment vessel 508 should form a vacuum seal with avacuum fixture (not shown) such that a pressure differential can formbetween the sample in the treatment vessel and the supplied vacuum. Oncethe vacuum is applied to the fixture, the pressure differential acrossthe upper member 530 will cause the upper member 530 of the treatmentvessel 508 to rupture and cause the lower member 550 to collapse intothe funnel 592. The lower member 550 should have sufficient strength sothat it does not rupture where it bridges the opening in the small endof the funnel 592. The pressure differential will cause the solidcontents 596 of the treatment vessel to be squeezed between the flexiblelower member 550 of the treatment vessel 508 and the relatively rigidfunnel 592. This causes fluid 542 to be expelled from the solidmaterials 596 and collected in the post-treatment vessel 598.

In certain other embodiments, a treatment vessel can be an ampoule,vial, pouch, bag, or envelope. These and other treatment vessels can beformed from such materials as polyethylene, polypropylene, poly(ethyleneteraphthalate) (PET), polystyrene, acetal, silicone, polyvinyl chloride(PVC), phenolic, glasses and other inorganic materials, metals such asaluminum and magnesium, and laminates such as polyethylene/aluminum andpolyethylene/polyester. Also, certain embodiments of a treatment vesselcan be made by vacuum forming, injection molding, casting, and otherthermal and non-thermal processes. In embodiments where samples flowthrough the sonic energy, capillary tubes, etched channels, and conduitsmay be the sample holder during treatment as the sample flows through astructure. Additionally, free-falling drops, streams, non-moving freevolumes, such as those in gravity less than one g, or a layer in adensity gradient can be treated directly.

II. Materials for Treatment

A. Biological Materials

Many biological materials can be treated according to the invention. Forexample, such materials for treatment include, without limitation,growing plant tissue such as root tips, meristem, and callus, bone,yeast and other microorganisms with tough cell walls, bacterial cellsand/or cultures on agar plates or in growth media, stem or blood cells,hybridomas and other cells from immortalized cell lines, and embryos.Additionally, other biological materials, such as serum and proteinpreparations, can be treated with the processes of the invention,including sterilization.

B. Binding Materials

Many binding reactions can be enhanced with treatments according to theinvention. Binding reactions involve binding together two or moremolecules, for example, two nucleic acid molecules, by hybridization orother non-covalent binding. Binding reactions are found, for example, inan assay to detect binding, such as a specific staining reaction, in areaction such as the polymerase chain reaction where one nucleotidemolecule is a primer and the other is a substrate molecule to bereplicated, or in a binding interaction involving an antibody and themolecule it binds, such as an immunoassay. Reactions also can involvebinding of a substrate and a ligand. For example, a substrate such as anantibody or receptor can be immobilized on a support surface, for use inpurification or separation techniques of epitopes, ligands, and othermolecules.

C. Chemical and Mineral Materials

Organic and inorganic materials can be treated with controlled acousticpulses according to the methods of the invention. The sonic pulses maybe used to comminute a solid material, particularly under a feedbackcontrol regime, or in arrays of multiple samples. As with biologicalsamples, individual organic and inorganic samples in an array can betreated in substantial isolation from the laboratory environment. Besidealtering their physical integrity, materials can be dissolved in solventfluids, such as liquids and gasses, or extracted with solvents. Forexample, dissolution of polymers in solvents can be very slow withoutstirring, but stirring multiple samples with current methods isdifficult and raises the possibility of cross-contamination betweensamples. However, stirring of multiple samples withoutcross-contamination between samples can be accomplished with apparatusand methods of the invention.

III. Treatment Applications

A. Altering Cell Accessibility

Sonicators can disrupt cells using frequencies around 20 kHz. It isgenerally thought there are two ways in which ultrasound can affectcells, namely by heating and by cavitation, which is the interaction ofthe sound wave with small gas bubbles in the sample. Heating occursprimarily due to absorption of the sound energy by the medium or by thecontainer. For dilute aqueous systems, it is absorption by the containerthat is a main source of the heating. Heating is not desirable in sometreatment applications, as described herein. The heating associated withthe compression and cooling associated with the rarefaction of a soundwave is relatively small, even for intense sound.

According to the invention, controlled sonic pulses in a medium are usedto treat a sample containing biological material. The pulses can bespecifically adapted to preferentially interact with supporting matricesin a biological material, such as plant cell walls or extracellularmatrices such as bone or collagen, thereby lessening or removing abarrier function of such matrices and facilitating the insertion ofextracellular components into a cell. In this application, the cell isminimally altered and cell viability is preserved. These pulses can becaused by shock waves or by sound waves. The waves can be createdexternal to the sample, or directly in the sample, via appliedmechanical devices. In experiments where thermal effects are negligible,there typically is no lysis, unless cavitation is present. Other modesof sonic energy can have different effects than disrupting a matrix andcan be used either with pre-treatment, with disrupting sonic energy, orby themselves. For, example the conditions to disrupt a matrix can bedifferent from those to permeabilize a cell membrane.

There are many possible mechanisms by which cavitation may affect cellsand there is no consensus as to which mechanisms, if any, dominate. Theprinciple mechanisms are thought to include shear, microjets, shockwaves, sonochemistry, and other mechanisms, as discussed more fullybelow.

Shear: Significant shear forces are associated with the violent collapseof bubbles. Because cell membranes are sensitive to shear, it is thoughtthat cavitation may permeabilize cell membranes. In some cases, themembrane is apparently permeable for only a short time, during whichmolecules may be passed into or out of the cell. In other cases the cellmay be lysed.

Microjets: Bubbles undergoing a violent collapse, particularly near aboundary, such as a container wall, typically collapse asymmetricallyand generate a liquid jet of fluid that passes through the bubble andinto the boundary. The speed of this jet has been measured to behundreds of meters a second and is of great destructive power. It mayplay a major role in the destruction of kidney stones by acoustic shockwaves and may be a possible way of destroying blood clots.

Shock wave: Collapse of a bubble spherically can generate an intenseshock wave. This pressure can be thousands of atmospheres in theneighborhood of the bubble. The compressive stress of the shock wave maybe strong enough to cause cellular material to fail.

Sonochemistry: The pressure and temperatures in the bubble during aninertial collapse can be extraordinarily high. In extreme examples, thegas can be excited sufficiently to produce light, termedsonoluminescence. Although the volume is small and the time durationshort, this phenomenon has been exploited to enhance chemical reactionrates. The production of free-radicals and other sonochemicals may alsoaffect cells.

Other: Other factors also may be involved. Vessel walls may contributecavitation nuclei. A plastic vessel with an aqueous fluid may result ina standing wave field due to internal reflections, as a result ofimpedance mismatches between the fluid and the vessel walls. Examples ofsonolucent materials are thin latex and dialysis tubing. Tube rotationstudies performed on continuous wave dosage with unfocused ultrasonicsindicate that rotation has a significant effect on hemolysis. When cellcontents were mechanically stirred during insonation, the cell lysisincreased. These effects may be due to viscosity gradients set-up withinthe unfocused ultrasound field that block energy transmission.

Cellular lysis also can be aided by the addition of ultrasound contrastagents, such as air-based contrast agents or perfluorocarbon-basedcontrast agents. An example of an air-based contrast agent is adenatured albumin shell with air such as Albunex, available fromMallinckrodt, St. Louis, Mo., and an example of a perfluorocarbon-basedcontrast agent is a phospholipid coating with perfluoropropane gas suchas MRX-130, available from ImaRx Pharmaceutical Corp., Tucson, Ariz.

Air bubbles can block or reflect energy transmission. Interfaces betweenair and water result in efficient reflection of an incident ultrasoundfield.

The treatment dose is a complex waveform. Sections, or components, ofthe waveforms can have different functions. For example, the waveformcan have three components involved with sample mixing, samplelysis/disruption, and sample cooling.

In other current methods, sonolytic yield activity decreases withincreasing cell concentrations in in vitro systems that are treated withcontinuous ultrasound waves. In contrast, methods according to thepresent invention disrupt tissue structures with a complex waveform ofhigh intensity focused ultrasound, to avoid this problem.

Mixing can be an important, because it allows bubbles that may have beendriven by radiation forces to the edges of the vessel chamber to bebrought into contact with the cell or tissue membranes. This mixingpromotes inertial, transient acoustic cavitation near the cell walls,resulting in cellular lysis.

The acoustic dosage received by a sample can be likened to a radiationdosage received by a sample. In each case, a cumulative effect of theabsorbed energy dose is observed. A computer-controlled positioningsystem can control the cumulative energy dosage that each samplereceives. For example, a software program in the computer can activelycontrol the cumulative energy dosage by treating the sample until thesystem reaches a particular set-point, pausing energy application orotherwise allowing the sample to reequilibriate, and reinitiating energyapplication to allow a sample to receive a higher cumulative dose whilemaintaining semi-isothermal conditions, such as a 1 to 2 degreeCentigrade temperature rise during exposure, than would otherwise bepossible by continuous sonic energy application. This type of systemenables high energy to be introduced into a sample while maintainingthermal control of the process.

B. Extracting

In a variation of the method to alter cellular accessibility describedabove, controlled pulses in a medium can be used to treat a samplecontaining biological material to extract a fraction or fractions of thebiological material. The pulses are specifically adapted topreferentially interact with supporting matrices, such as plant cellwalls or extracellular matrices such as bone or collagen, or materialshaving differences in rigidity or permeability in a biological material,thereby lessening or removing a barrier function of such matrices ormaterials. These pulses can be caused by shock waves or by sound waves.The waves can be created external to the sample, or directly in thesample, via applied mechanical means.

Using sound energy, as opposed to laser or other light energy to disrupta biological object, can be useful. Sound is a direct fluctuation ofpressure on the sample. Pressure is a physical quantity and the measureof uniform stress defined as the force per unit area. The stress actingon a material induces strain which changes dimensions of the material.The two main types of stress are a direct tensile or compressive stressand shear stress. In general, the more brittle the material, the greaterthe disruptive effect of an abrupt, local increase of otherwise uniformstress. Such a local stress can be created by some geometric change at asurface or within the body of the sample. For example, biological tissuefrozen at −70° C. may be more prone to stress fracture than at 4° C. Inaddition, a sharper change in geometric or material properties tends tocause a greater stress concentration, which in turn can yield a greaterdisruption. Sound waves may be focused. In contrast, the energytransferred from a light source such as a laser to a sample iselectromagnetic radiation that induces non-ionizing molecular vibrationsand breaks chemical bonds by ionizing. Mechanical stress on objectslarger than molecules generally cannot be readily caused byelectromagnetic waves, except via destructive local heating.

The supporting matrix of a biological sample can be disrupted withoutdisrupting one or more selected internal structures of the cellscontained within the matrix. Representative examples of such samplesare: i) bone, in which a rigid matrix contains living cells of interest;ii) mammalian tissue samples, which contain living cells embedded in amatrix of elastic connective tissue and “glycocalyx” or intercellularmatrix; and iii) plant tissues, such as leaves, which contain cells in amatrix of cellulose, often crosslinked with other materials, of moderaterigidity. Virtually all living cells are gelatinous in texture, and canbe deformed to some extent without rupture or internal damage. Matrices,in contrast, are designed to support and protect cells, as well as toachieve other biological functions. In the three examples above, thematrices of bone and leaves are designed to provide rigidity to thestructure, while the support of most collagenous matrices has a stronglyelastic character. Thus, different protocols for example, amplitude,duration, number of pulses, and temperature of sample, may be used todisrupt different matrices by mechanical means without damaging thecellular material.

A bony matrix is both more rigid and denser than the cells it contains.Bone is vulnerable to shock waves, both because the calcified matrixwill absorb the waves more efficiently than will the cells, and becausethe calcified matrix is weak under extensional strain, and thereby canfragment at stresses which will not damage the softer cells. Similarconsiderations apply to leaf matrix, although the contrast in densityand modulus is less. In either case, a pulse, preferably a shock wave,is applied at an amplitude which is sufficient to fatigue the matrixcomponents while remaining below the amplitude required to damage thecells. This intensity is determined readily for a particular type ofsample by minimal routine experimentation. In such experiments, theamplitude of each pulse applied to the sample, singly or in a train ofpulses, is varied to obtain the maximum rate of degradation of thematrix consistent with retention of the viability of the cells withinthe matrix. These parameters can be measured readily. For example,matrix degradation can be measured by variation in the compressivemodulus of the sample, while cell integrity is measured by dye exclusionfrom cells extracted from the matrix, such as, for bone,demineralization and treatment with collagenase. In the case of a moreelastic tissue, such as connective tissue, which is cross-linked but hasa high extension to break, the pulses are selected to excitepreferentially vibrational modes in the matrix in contrast to the cells.This can be done by selecting one or more frequencies of sound waves atwhich the relative absorptiveness of the matrix and the cells aremaximally different. Such frequencies are determined readily by routineexperimentation. A sequence of pulses may be required to differentiallyfatigue the matrix. The length of the pulses and the interval betweenthem are adjusted so that the degree of heating of the sample does notcause loss of integrity of the cells, and particularly of the criticalcomponents which are to be isolated.

Three areas to optimize for extraction are treatment waveform, mixingwaveform, and positioning or dithering. One method to determine theappropriate treatment and positioning parameters for a target sample forextraction purposes is described below.

First, a solid sample is placed in a volume of liquid in about a 1:1ratio (weight/volume), in a treatment vessel. For example, 0.25 ml ofmethanol is added to 0.25 gm of leaf tissue in a 0.5 ml treatmentvessel. A single sample is placed within the focal zone of the sonicapparatus. Without using the treatment protocol, the mixing waveform isadjusted to provide “stirring” of the sample at the lowest amplitude,fewest cycles/burst, and lowest duty cycle. After the mixing waveformprotocol is defined, the disruption treatment waveform is adjusted byimmobilizing the target sample in the focal zone such that there is nomixing and no sample movement, such as dithering. Using a sonic energysource such as a piezoelectric transducer, the sample is subjected to aminimum number of cycles per burst, for example, three. For extractionpurposes, the amplitude is initially used with a nominal 500 mV setting.A portion of the sample is treated and inspected under a microscope forsigns of membrane disruption. Such inspection can be done in conjunctionwith dyes that stain intracellular organelles. The number ofcycles/burst is then increased until a particular desired tissuedisruption level is achieved in the immobilized portion of tissue. Witha fresh sample, and with a 1:1 ratio of tissue to liquid, thetemperature of the sample is monitored during a million cycle totaltreatment with an infra-red sensor directed to the top of a thinpolyethylene film covering the sample vessel. The duty cycle is adjustedto keep the temperature within predefined ranges, such as 4° C. within+/−2° C.

Once these treatment parameters are discerned for a particular sample, acontrol unit can be programmed with these data in order to controltreatment of other samples of the same or similar biological type.Alternatively, such information can preprogrammed in the control unit,and an apparatus user, through a user input interface, can designate thebiological material type to be treated such that the controller thenruns through the predetermined treatment cycle. Other information can beempirically determined for optimal treatment of a particular biologicalmaterial in a manner similar to that described above. For example,parameters such as treatment waveforms, mixing waveforms, and samplepositioning can be discerned. These parameters can vary depending uponthe particular biological material, the particular liquid that surroundsthe sample, and/or the particular treatment vessel used duringtreatment.

C. Introducing a Molecule Into or Removing a Molecule From a Cell

Once a sample having a matrix has been sufficiently weakened orattenuated, but not to the point where a substantial number of cellscontained within the matrix are killed or lysed, an exposed target cellor cells become amenable to insertion of exogenous molecules bytechniques such as transfection or transformation. With some matrices,it may be convenient to isolate the cells from the matrices and then totransfect the cells. In other cases, it will be preferable, particularlyin an automated system, to perform the transfection directly on thetreated tissue sample, using solutions and conditions adapted from knowntechniques. Alternatively, in situations where a cell to be treated isnot situated within a matrix, the cell can be directly treated accordingto the process below without having to pre-treat the matrix. While thetreatment below is described mainly for transfection, methods andapparatus according to the invention are equally applicable to atransformation process or other processes to introduce an exogenousmaterial into a permeabilized cell membrane.

In general, cool temperatures, less than 25° C., preferably less than1520 C., more preferably 4° C. or below, tend to minimize thedegradative effects of enzymes in the sample and thereby tend topreserve the integrity of biological components to be isolated. However,cells, especially mammalian cells, may maintain their viability betterat higher temperatures, such as 30 to 37° C. These temperatures alsoallow enzymes to be added to aid in the selective destruction of thematrix.

Alternatively, the sample temperature may be below 0° C. Except underspecial conditions, this will freeze the sample, or maintain it in afrozen state. Freezing can be advantageous if it enhances the disruptionof the matrix while allowing the cell to remain relatively intact. Forexample, ice crystals formed on freezing can be selectively largeroutside of cells. Since such crystals may tend to absorb acousticalenergy better than water, destruction of the matrix may be enhanced.While decreasing cell viability and integrity, such a procedure couldenhance the ease of transfection with exogenous material after thawingof the sample.

The waveforms used to alter the permeability of a cell are refineddepending on the particular application. Typically, the shock wave ischaracterized by a rapid shock front with a positive peak pressure, forexample about 100 MPa, and a negative peak pressure, for example aboutnegative 10 MPa. This waveform is of a few microsecond duration, on theorder of about 5 microseconds. If the negative peak is greater thanabout 1 MPa, cavitation bubbles may form. Cavitation bubble formation isalso dependent upon the surrounding medium. For example, glycerol is acavitation inhibitive medium; whereas, liquid water is a cavitationpromotive medium. The collapse of cavitation bubbles forms “microjets”and turbulence that impinge on the surrounding material.

Sound waves, namely acoustic waves at intensities below the shockthreshold, provide an alternative means of disrupting the matrix toallow access to the plasma membranes of the cells to allowtransformation. Such sound waves can be generated by any known process.As biological material is subjected to subzero temperatures, for exampleabout negative 5° C., most but not all of the water is in the solidphase. However, in certain biological tissues micro-domains of liquidwater still remain for several reasons, such as natural “antifreeze”molecules or regions of higher salt concentration. Therefore, as asample temperature is varied during the treatment with sound or shockwaves, microdomains of liquid water are able to form shock waves andinduce cavitation bubble formation and collapse, with the resultantshear stresses that impinge on surrounding tissues. Indeed, gradualalteration of the sample temperature can be desirable, as it providesfocused domains of liquid water for impingement on the surroundingmaterial. The waves can be applied to the samples either directly, aspiezoelectric pulses, or via an intervening medium. This medium may bewater, buffer, stabilizing medium for the target material to beisolated, or extraction medium for the target. An intervening mediumalso can be a solid, formed of a material which is intrinsically solid,or of a frozen solution. Waves also can be applied through a container,such as a microtiter plate.

The techniques useful for disrupting matrix structure can be adapted,and the improved technique can be used, to facilitate the incorporationof exogenous material into cells. The exogenous material may be DNA,RNA, other nucleic acid constructs, nucleic acid monomers, plasmids,vectors, viruses, saccharides, polysaccharides, amino acids, amino acidchains, enzymes, polymers, organic molecules, inorganic molecules,proteins, cofactors, and/or visualization reagents such as fluorescentprobes. In this application, shock waves or sonic waves are used toloosen the matrix, essentially as described above. However, theintensity of application of acoustic energy is kept sufficiently short,or below a critical energy threshold, so that cell integrity iscompletely maintained, as verified by a method such as dye exclusion.

At that point, or, optionally, previously, a solution or suspensioncontaining the material to be incorporated into the cells is added tothe sample. In one embodiment, the exogenous material is incorporatedinto the cells in a conventional manner, as is known in the art forcells with exposed plasma membranes. In another embodiment, acousticenergy is used to transiently permeabilize a plasma membrane tofacilitate introduction of exogenous materials into the cells. Theexogenous material may be present in the sample during the weakening ofthe matrix by acoustic energy. Even when the cells remain intact, asdetermined by dye exclusion or other viability measurements, the processof weakening the cell matrix by acoustic energy transiently destabilizesthe plasma membranes, increasing the uptake of exogenous macromoleculesand structures. If a further increase in the rate of incorporation isneeded, then the intensity or time of application of acoustic energy isslightly increased until the cell membrane becomes transientlypermeable. For example, a gentle pulse or wave is applied to themixture, with a predetermined amplitude. This amplitude can bedetermined readily in separate experiments on samples of the same typeto transiently make a plasma membrane of a cell type porous, in asimilar empirical manner to the steps described above for determining anappropriate treatment to disrupt a matrix. During the transient porousstate, exogenous materials diffuse into the cell and the materials aretrapped there once the sonic or shock pulse is removed.

A major advantage of these methods for transfection, or otherincorporation of exogenous material into living cells, is that themethods are readily amenable to scale-up, to automation, and to markedreduction in sample size and reagent volume. The wells of microplatescan be used for sonic treatment, transfection, and post-transfectiondemonstration of successful incorporation of the added material. Forexample, extracellular non-incorporated reagent, for example afluorescent material, can be inactivated by a material that does notpass the cell membrane, such as an enzyme, or certain hydrophilic oramphiphilic small-molecule reagents. Then the presence or absence of therequired material can be determined directly in the sample, for exampleby spectroscopy. Thus, the methods are adaptable to large scaleautomation, in large part because they do not require the isolation ofthe cells from their matrix.

The permeabilized cells can be transformed or transfected, usingtechniques known to those skilled in the art, for example,electroporation, vacuum transfection, or using viral vectors,agrobacterium, liposomes or other delivery vehicles, plasmids, or nakednucleic acids. The buffer conditions may be altered during the process.For example, the initial permeabilization may occur with chemicals toselectively alter the external cell wall, while during the nuclear wallpermeabilization step, other chemicals or biochemicals may be added toprompt selective uptake.

Additionally, with the process of permeabilization and with the mixingprofile, other techniques of gene transfer may be augmented. Examplesinclude, calcium phosphate coprecipitation, electroporation, andreceptor-dependent processes.

D. Mixing, Stirring, and Heating

In fluid samples, including powdered and granular media and gasses,sample mixing is conventionally performed by vortexing or stirring, orother methods such as inversion of a sample containing an air space, andshaking. Vortexing is essentially achieved by mechanical motion of theentire vessel while stirring involves mechanical contact of a drivendevice with a fluid. Stirring is accomplished with a variety of devices,for example with propellers, impellers, paddles, and magnetic stir bars.One problem with these methods is that it is difficult to increase theirscale in order to handle dozens or hundreds of sample vessels at once.Another problem with these methods is the difficulty of mixing multiplesamples while keeping the each sample substantially free fromcontamination. As described in more detail below with respect to FIGS.8-17, methods according to the invention can use sonic energy to mix asample while avoiding problems with contamination. More particularly,factors, such as focusing the sonic energy, as well as otherwisecontrolling an acoustic waveform of the sonic energy to be directed atone or more nucleation sites, and selectively locating nucleation sitescan be used to selectively mix a sample, for example, through acousticstreaming and/or microstreaming.

A fluid sample can be mixed controllably using the system describedherein. No direct contact between the material to be mixed and the sonicenergy source is required. However, in some embodiments, contact ispreferable. When the material to be mixed is in a treatment vessel, suchas a microplate, the treatment vessel itself is not necessarily touchedby the source and is typically coupled to the source by a fluid bath. Inother embodiments, the acoustic source is a microsource located within amicrodevice chamber.

In certain embodiments, a treatment process for sample mixing in atreatment vessel can be summarized as follows. First, a sample istreated with sonic energy at a relatively high first treatment power inorder to heat the sample by absorption of acoustic energy. Second, thesample is mixed at a second sonic energy power, which may be the same orlower than the first treatment power, to cool the sample back to itsoriginal temperature by forcing convection through material in thetreatment vessel, which can be in contact with a fixed-temperature bathor reservoir.

In some embodiments, a source of focused ultrasonic waves is used. Thesource is mounted in a water bath or equivalent, which can providetemperature control. The microplate, with samples in the wells, ispositioned so that the focus of the beam is within the well. The plateis positioned so that the bottoms of the wells are in contact with orimmersed in the water or other fluid in the bath. Then, a burst ofultrasonic energy is applied to the well. This burst will cause stirringin the well, by formation of a convection cell. The stirring is easilyvisualized by adding particulate material to the wells, or by adding adye in a denser or lighter solution.

It is possible to select a sound field which will stir all of the wellsof a plate at one time. In one embodiment, a substantially uniform fieldis projected to the plate by a source, which preferentially excites thebottoms of the wells. This excitation in turns drives convective flow ineach of the wells. In other embodiments, nucleation features are locatedin the plate proximate to each of the wells, or even inside the wells,to enable mixing to occur at lower energy levels.

In any embodiment, it can be useful to move the sample treatment vessel,such as by “dithering” the plate or well being treated relative to thesource. Dithering, as used in optics and in laser printing, is a rapidside to side two or three dimensional movement of the energy sourceand/or the target. Dithering, or other types of motion, can even outvariations in source intensity due to variations in the emitted sonicenergy or the location of the sample with respect to the source.Dithering can also prevent particulates from accumulating at the wall ofthe well. Fluid control features, such as mixing, are discussed below infurther detail with respect to FIGS. 8-17.

E. Enhancing Reactions and Separations

In certain embodiments, temperature, mixing, or both can be controlledwith ultrasonic energy to enhance a chemical reaction. For example, theassociation rate between a ligand present in a sample to be treated andan exogenously supplied binding partner can be accelerated. In anotherexample, an assay is performed where temperature is maintained andmixing is increased to improve association of two or more moleculescompared to ambient conditions. It is possible to combine the variousaspects of the process described herein by first subjecting a mixture toheat and mixing in order to separate a ligand or analyte in the mixturefrom endogenous binding partners in the mixture. The temperature,mixing, or both, are changed from the initial condition to enhanceligand complex formation with an exogenously supplied binding partnerrelative to ligand/endogenous binding partner complex formation atambient temperature and mixing. Generally, the second temperature and/ormixing conditions are intermediate between ambient conditions and theconditions used in the first separating step above. At the secondtemperature and mixing condition, the separated ligand is reacted withthe exogenously supplied binding partner.

Polymerase Chain Reaction (“PCR”) Thermal Cycling

One of the bottlenecks of the PCR technique is cooling time. The heatingcycle is rapid; however, cooling is limited by convection. Even inbiochip formats, in which DNA or another target molecule is immobilizedin an array on a microdevice, there is no “active” cooling process.However, certain embodiments of the invention can be used to overcomethis bottleneck.

In certain embodiments, a treatment process can be used to both heat andcool the sample rapidly with little overshoot from a baselinetemperature at which the primer and target to be amplified anneal. Theprocess can be summarized as follows. A sample is treated withrelatively high power sonic energy such that the sample absorbs sonicenergy and is heated. Then, the sample is mixed at low power to cool thesample by forcing convection, which may be accomplished in conjunctionwith a cool water bath. In some embodiments of the apparatus, the systemis a “dry top” system, that is, a system in which a microplate,typically with its top temporarily sealed with plastic film, floats onor is partially immersed in a controlled-temperature bath. In thisarrangement, the PCR reaction may be monitored in real-time fortemperature, using, for example, an infra-red detection probe, and forreaction products by examining the incorporation of fluorescent dyetagged nucleic acid probes into the PCR product. This “dry top” systempermits real-time analysis and control of the process. Information fromthe temperature sensor can be used in a feedback loop to control theduty cycle of the acoustic input, such as the number of bursts/second,or otherwise control the amount of heating. Also, fluorescence from anintercalated probe can provide a computer with information on whichwells have reached a certain point in the reaction, such as when aparticular level of fluorescence is sensed, allowing, for example, thecomputer to control application of sonic energy or sample location suchthat certain wells are skipped in the processing cycle until other wellshave attained the same point in the reaction or that certain wells arenot processed further.

F. Purification, Separation, and Reaction Control

Focused sonic fields can be used to enhance separations. As notedelsewhere, sonic foci can be used to diminish or eliminate wall effectsin fluid flow, which is an important element of many separationprocesses, such as chromatography including gas chromatography, sizeexclusion chromatography, ion exchange chromatography, and other knownforms, including filed-flow fractionation. The ability to remotelymodulate and/or reduce or eliminate the velocity and concentrationgradients of a flowing stream is applicable in a wide variety ofsituations.

Sonic fields also can be used to minimize concentration polarization inmembrane processes, including particle classification, filtration offine particles and colloids, ultrafiltration, reverse osmosis, andsimilar processes. Concentration polarization is the result of thetendency of filtered material to be present at high concentration in alayer on the filter. This layer has a low fluid concentration and, thus,diminishes the rate of filtration as the filtered solution becomes moreconcentrated, or as the layer thickens. This layer can be stirredremotely by focused sonic energy of low to moderate intensity. Flowrate, thus, can be enhanced without significant cost in energy ormembrane life.

Such sonic energy fields can be used to enhance reaction rates in aviscous medium, by providing remote stirring on a micro scale withminimal heating and/or sample damage. For example, some assays rely onthe absorption of analytes by reagents, such as antibodies, which arebound to macroscopic particles. In a viscous fluid to be analyzed, suchas sputum or homogenized stool, the ability to stir such a sampleremotely, aseptically, and essentially isothermally can significantlydecrease the time required to obtain equilibrium of the analyte with thereagents on the particle.

Likewise, any bimolecular (second-order) reaction where the reactantsare not mixed at a molecular scale, each homogenously dissolved in thesame phase, potentially can be accelerated by sonic stirring. At scaleslarger than a few nanometers, convection or stirring can potentiallyminimize local concentration gradients and thereby increase the rate ofreaction. This effect can be important when both reactants aremacromolecules, such as an antibody and a large target for the antibody,such as a cell, since their diffusion rates are relatively slow anddesorption rates may not be significant.

These advantages may be realized inexpensively on multiple samples in anarray, such as a microtiter plate. The use of remote sonic mixingprovides a substantially instantaneous start time to a reaction when thesample and analytical reagents are of different densities, because insmall vessels, such as the wells of a 96 or 384 well plate, littlemixing will occur when a normal-density sample (about 1 g/cc) is layeredover a higher-density reagent mixture. Remote sonic mixing can start thereaction at a defined time and control its rate, when required. Thestepping and dithering functions allow multiple readings of the progressof the reaction to be made. The mode of detecting reaction conditionscan be varied between samples if necessary. In fact, observations bymultiple monitoring techniques, such as the use of differing opticaltechniques, can be used on the same sample at each pass through one ormore detection regions.

G. Further Uses For Remotely Actuated and Controlled Solution MixingWith Sonic Energy

Control of sonic energy emission, sonic energy characteristics, and/orlocation of a target relative to sonic energy also can be used to pumpand control the flow rate of liquids, especially in capillaries; enhancechemical reactions, such as enhancing second-order reaction rates;increase effective Reynolds number in fluid flow; and control thedispensing of semi-solid substances.

By focusing sonic energy and positioning it near a wall of a vessel, awall of a tube, or another discontinuity in a fluid path, many localdifferences in the distribution of materials within a sample and/orspatially-derived reaction barriers, particularly in reactive andflowing systems, can be reduced to the minimum delays required formicroscopic diffusion. Put differently, enhanced mixing can be obtainedin situations where imperfect mixing is common. The range of thesesituations is illustrated below.

Control of Flow Rates of Fluids

Miniaturization of analytical methods, such as analysis on a chip,require concomitantly miniature capillary-sized dimensions for fluidflow paths. Sonic excitation provides a convenient, simple, and sterilemanner to accelerate flow in capillaries. During excitation, the fluidis locally turbulent, and so flows more readily. By selective or timedlocal sonic excitation, optionally controlled with a feedback loop, therate of flow through complex microfluidic paths can be remotelymanipulated in a controlled manner. As described below with respect toFIGS. 6 and 7, methods of the invention can not only be used to increaseflow, but can also be used to inhibit flow.

Increase of Effective Reynolds Number in Fluid Flow

At low Reynolds numbers, the velocity profile of laminar fluid flow in apipe or other conduit is approximately parabolic. Fluid at the center ofthe pipe is flowing significantly faster than fluid near the wall.Therefore, conversion of fluid carried in the pipe from one fluid toanother is quite slow, and, in principle, infinitely slow.

This effect effectively vanishes at higher Reynolds numbers becauseturbulents mix the fluid at the center with fluid at the periphery veryrapidly, so that composition differences are rapidly eliminated.However, there are significant disadvantages to operating a fluidconduit under turbulent conditions, including high backpressure andcorrespondingly high energy expenditure.

If sonic energy is focussed in, on, or near the wall of the pipe, nearthe fluid/wall boundary, then local turbulence can be obtained without ahigh rate of bulk fluid flow. Excitation of the near-wall fluid in acontinuous, scanned, or burst mode can lead to rapid homogenization ofthe fluid composition just downstream of the excited zone. This willsharpen the front between any two fluids passing through a pipe insuccession. Additionally, using the further methods described in FIGS.8-17, cavitation features can be located on or in the wall of a pipe tofacilitate homogenization of the fluid flow, with the use of unfocussedacoustic energy.

This effect is useful in several areas, including chromatography; fluidflow in analytical devices, such as clinical chemistry analyzers; andconversion of the fluid in a pipeline from one grade or type to another.Since most of the effect occurs in a narrow zone, only a narrow zone ofthe conduit typically needs to be sonically excited. For example, insome applications, the focal zone of the sonic energy can be the regionclosest to a valve or other device which initiates the switch ofcomposition. In any of these applications, feedback control can be basedon local temperature rise in the fluid at a point near to or downstreamof the excitation region.

Enhancement of Second-Order Reaction Rates

Microsonication can be used to speed up, or to homogenize, the rate ofchemical reactions in a viscous medium. The flow of individualmolecules, and of heat, is generally slower in a more viscous medium.For example, it is more difficult to mix molasses with water than to mixvinegar with water. Similarly, in an aqueous solution, it becomesincreasingly difficult to maintain the rate at which soluble monomersundergo a polymerization reaction, forming a soluble polymer, as themolecular weight of the polymer increases with each added monomer,because the viscosity of the solution increases.

Mixing of molasses and water with a stirrer is simple, but not easilysterile, and a polymer can be degraded by shear caused by stirring witha stirrer. Focussed sonication can readily mix pre-sterilized liquids ina remote manner without contamination. Focused sonic energy also can mixpolymerizing materials without application of macroscopic shear, and socan minimize shear degradation of the formed polymer. Similarly, apolymerase chain reaction can be accelerated by brief pulses of sonicenergy, or by longer pulses which also provide the desired temperatureincreases, to prevent the retardation of the reaction due to localdepletion of the nucleotide triphosphate monomers. As described belowwith respect to FIGS. 8-16, similar results can be achieved withunfocussed sonic energy by selectively locating nucleation features toenable cavitation at lower energies.

Controlled Dispensing of Semi-Solid Substances

Highly viscous liquids, including materials which effectively act assolids or near-solids, can flow at an increased rate when sonicallyexcited by a remote or local sonic source. This excitation may be underfeedback control. This effect can be caused by local reduction ofimpedance to flow by walls of a conduit, as described above, and bylocal heating from sonic energy input. As a simple example, theeffective viscosity of an ink jet ink, and thus the rate of itsdelivery, can be controlled by focused, localized sonic energy delivery.Analogous uses are possible wherever the viscosity of a fluid, includinga semi-solid or a solid capable of melting, is significant. Likewise,flow of particulate materials in a fluid where the particles areinsoluble in the fluid can be selectively stimulated to occur, or beaccelerated, with focussed, controlled sonic waveforms.

VI. Further Apparatus and Methods for Employing Nucleation Features toControl Fluid Movement

As described briefly above, the acoustic source 230 and controller 410may be fabricated integrally with a microdevice containing a liquid tobe mixed or caused to flow. Alternatively, the controller 410 may befabricated as a separate remotely located component and communicativelycoupled (for example, via an electrical or optical interface) to thesource 230. In a further illustrative embodiment, the acoustic source230 may also be fabricated separately and located remotely from themicrodevice. In such an embodiment, the acoustic source 230 couples tothe microdevice for example, by way of a solid, liquid, gel, vapor orgas couplant.

In one embodiment, the source 230 is fabricated to be located inside amicrochamber of a microdevice, and is in direct contact with the fluidto be controlled.

According to the illustrative embodiment, the acoustic field energy 240is sufficiently intense to at least form a bubble in a target zone 800(which may correlate with the focal zone of the source if the source isfocused). In another embodiment, the acoustic field 240 is sufficientlyintense to alternate between bubble formation and bubble decay orcollapse in the target zone 800. At still higher energies the acousticfield 240 causes bubble formation, streaming, and collapse at a locationdifferent from the nucleation site; this is preferred for some types ofmicrofluidic applications, such as mixing in a fluid device.

As described briefly above, the methodology of the invention includestwo important aspects. In a first aspect, the invention directs theacoustic field 240 at through, for example, blocking, focussing, and/orreflection, at nucleation features to control fluid movement. In asecond aspect, the invention provides nucleation features at selectedlocations and then interacts either focussed or unfocussed acousticenergy with the selectively located nucleation features to control fluidflow.

FIG. 8 is a conceptual diagram 800 depicting an illustrative life cycleof a bubble formed on a surface 802 having a crevice or surface defect804, which acts as a nucleation feature, according to an illustrativeembodiment of the invention. When acoustic energy is directed toward thefeature 804, cavitation occurs in a controlled, beneficial manner.Typically, the acoustic energy 240 required for cavitation is lower withcavitation promoters, such as the nucleation feature 804, than withoutpromoters. Illustratively, and as shown at 807, in response to thesurface 802 being immersed in a solution, such as an aqueous solution506, using, for example, a mechanism such as is depicted in FIG. 7, agas or vacuum pocket 808 forms and creates a liquid-gas (vapor)interface in the feature 804. As shown at 809, in response to the pocket808 being excited by the acoustic energy 240, the pocket 808 grows toform a bubble 810 and locally displaces the fluid 806. In response torepeated cycles of negative and positive pressures from the acousticfield 240 and to increasing the energy of the field 240 above athreshold energy level, cavitation occurs. By alternatively providingand removing the acoustic field 240, the bubble 810 can be made toalternatingly increase (812) and decrease (814) in diameter. Thealternating rarefaction (812) and compression (814) process causeslocalized flow in the fluid 806 proximate to the feature 804, and thuscreates a localized micromixing action. Depending on characteristics(e.g., frequency duty cycle amplitude, etc., discussed in detail above)of the acoustic field 240, the bubble 810 can collapse either slowly orabruptly. As shown at 816, in response to discontinuing the acousticfield 240, the bubble 810 dissipates back to the pocket 808, and thusceases the micromixing action. According to the illustrative embodiment,nucleation sites can be selective located anywhere that aids fluidcontrol. It should also be noted that exposure to the acoustic field 240may also cause heating of the device 802 and the fluid 806, and that, asdescribed above, the effects of the field 240 can be changed by varyingcharacteristics of the acoustic field 240. Also, the acoustic field 240may or may not be particularly directed at the feature 807. Also, thefeature 807 may be selectively located or may naturally occur as aresult of the fabrication of the surface 202.

The acoustic field 240 may be a traveling wave field or a standing-wavefield. The frequency and other parameters of the acoustic field 240 maybe varied to achieve and control the desired effects and minimizeundesired effects. As described with respect to FIGS. 1-7, the field 240may be moved in a regular pattern relative to the microdevice containingthe fluid to be mixed to achieve rapid results over the entire volume ofthe microdevice, or may be moved in a random or pseudo-random pattern totreat the microdevice in a stochastic manner. The frequency of theacoustic field 240 may be varied over time. For instance, in a standingwave field, the frequency may be swept upward during a tone burst tomove the interference pattern closer to the axis and thus sweep materialto the center. Or, a series of tone bursts at increasing frequencies maybe applied to achieve the same effect. In another example, a highfrequency, such as 1.1 MHz, may be applied to generate bubbles in thedevice and a lower frequency may then be applied to cause them tocollapse.

For example, if in a particular apparatus, a cavitation promotingfeature is excited by 1.1 MHz acoustic energy produced by a 100 mVexcitation of the transducer, then a pulse of 10 cycles will generate abubble, producing waves in the solution tending to mix it locally. Ifthe energy 240 is then turned off for 10 cycles, the bubble willcollapse. Alternatively, the bubble may oscillate at an energy less thanthat required to initiate a bubble, such as 25 mV. (Note: these voltagesare for a particular apparatus and are only being used illustratively.)Which mode of bubble removal is best will depend on the particularapparatus and purpose, and can be determined experimentally if required.

A microdevice, containing fluid to be mixed may or may not be near or atthe focus of a focused acoustic field 240. If the microdevice is in ornear the focal plane of a focused acoustic field 240, the intensitygradient in the plane of the microdevice will be at a maximum. If themicrodevice is moved into the near field or the far field of theacoustic field 240, the intensity gradient in the plane of themicrodevice will be lowered. The microdevice may be moved along the axisof the field 240 during or between treatments to effect mixing withinthe microdevice.

Illustrative experiments have employed both focused and unfocussedacoustic fields 240 at a certain frequency (about 1.1 MHz) orientedperpendicular to the plane of the microdevice. However, similar effectsmay be obtained with an unfocused field 240 whose direction ofpropagation is tilted relative to the plane of the microdevice or at adifferent or time-varying frequency. In addition, two or more acousticfields 240 may be caused to interact or interfere in the microdevice tocause mixing.

Referring briefly to FIGS. 10 and 11, in active site arrays, such as theDNA arrays depicted in FIG. 11 and discussed below, the hybridizationchamber, such as that depicted at 1004 in FIG. 10, is typically a planardevice. The apparatus of the invention may be arranged such that theacoustic field 240 can irradiate the device from either the base(bottom) 1016 side or the cover (top) 1014 side. Alternatively, theacoustic source 230 may be integrally fabricated in an internal wall ofthe chamber 1004. The features, such as 1006 a and 1006 b, that promotemixing may be located on either inside surface of the microdevice 1002such that they are on either the near side or the far side relative tothe acoustic field 240. As stated above, the axis of the acoustic field240 may be perpendicular to the microdevice, parallel to the plane ofthe microdevice, or at an angle relative to the plane of themicrodevice. Also, the acoustic field 240 may or may not be particularlydirected at the nucleation features 1006 a and 1006 b.

Moving the microdevice 1002 relative to the acoustic field 240 during orbetween treatments can offer several advantages. Dithering (smallvariations in position around a center point) in the X-Y plane duringtreatment changes the intensity gradient in the plane of the microdevice1002 and causes flow patterns to change direction and intensity. Thiscauses more uniform mixing over larger areas. This may also cause bettermixing in the corners of a square or rectangular hybridization chamber.Motion in the z (axial) direction causes changes in the interferencepattern in a standing wave field, and may be used to break upaggregations of particles that may have formed.

In the illustrative embodiment, the cavitation promoting features may bea single feature, such as a pit, or a multitude of features such as afield of pits or a hatch pattern of lines or grooves. In the case of asingle feature or a few features spaced such that only one is active ata time, the device may be moved relative to the acoustic field 240 suchthat the flow pattern generated by the feature will vary with time.Specifically, the cavitation promoting feature may be dithered aroundthe focal zone of a focused acoustic field 240 such that the flowpattern rotates around the feature. This is analogous to watering afield with a processing sprinkler head: the flow is directed in a singledirection at any one point in time, but the flow direction is variedover time to water a large area uniformly.

As mentioned above, turning on the acoustic field 240 causes mixing.Turning it off causes mixing to stop. The on and off intervals may becontrolled to maximize mixing while allowing enough quiescent time orresidence time or contact time for binding reactions or other chemicalreactions to take place before the reactants are separated by anothermixing event. The temperature of the microdevice may be cycled up anddown in coordination with the mixing intervals to influence theoccurrence of reactions or the rate of reactions within the microdevice.

The cavitation threshold is the acoustic intensity at which cavitationbegins at a particular region in a fluid. The cavitation threshold isinfluenced by several factors including isotropic pressure, dissolvedgas content and fluid composition as well as the presence or absence ofnucleation features. The acoustic field 240 may be operated above thecavitation threshold to cause beneficial cavitation effects in amicrodevice or may be operated below the cavitation threshold to obtainother effects not related to cavitation, such as heating.

As discussed above, cavitation can be detected by means of a passivecavitation detection transducer and the appropriate electronics 700 orany of several other well known mechanisms. This detection mechanism maygenerate an error signal for use in a feedback control mechanism tomodulate the acoustic field 240. The term “passive” implies that thedetected signal is generated by cavitation. An “active” cavitationdetection system interrogates the region of interest with an externalenergy source (e.g., a laser light source) that is modulated bycavitation bubbles in the device. In a preferred embodiment, a passivecavitation receiver transducer is positioned confocally with a sourcetransducer of the acoustic field, such as the source transducers of FIG.6, or the source 230 of FIG. 1. The receiver transducer should have afrequency pass band higher than, and not substantially overlapping, thepass band of the source transducer. Cavitation signals of interest aregenerally higher than the source 230 frequency. Other embodimentsinclude having overlapping pass bands combined with suitable filters orsignal processing to suppress the source frequency from the receivertransducer signal. Another embodiment may involve using the sourcetransducer itself as a receiver for cavitation detection. There areseveral methods of passive and active cavitation detection that aredescribed in the literature. Any or all of these may be suitable fordetecting cavitation in conjunction with the present invention.

In another illustrative embodiment, cavitation promoting nucleationfeatures and texture details are on the exterior of a sample chamber toaffect the acoustic field within the chamber to promote bubble formationand streaming in the areas to be mixed in the interior.

According to the illustrative embodiments, it is recognized thatparticles in a solution can lower nucleation thresholds similarly to“features” or “textures” on the walls of a chamber or passage. Hence, ifthe particles constitute a solid phase resin having reticulatedsurfaces, such as are used to bind DNA and other biomolecules forbiochemical processes, not only will treatment with a moving acousticfield as described above result in increased mixing and enhancement ofdiffusion limited reactions, but fluid circulation within the particlesthemselves may be enhanced by pulsating gas bodies on or within theparticles. Similarly, the size, distribution of the particles, and thesurface features of the particles, as well as their surface wetability,may be optimized for mixing in a particular acoustic field based on thefrequency, intensity or other characteristics of the acoustic field 240.

FIG. 9 is a conceptual diagram 900 depicting the stages of nucleation ina microcavity according to an illustrative embodiment of the invention.As shown, the substrate 902 includes a microcavity 904. A naturallyoccurring or intentionally created crevice 906 is located within themicrocavity 904. In response to being brought in contact with a liquid908, the crevice 906 nucleates a small bubble 908. As shown at 910, 912and 914, the bubble 908 grows in diameter with each pressure cycle fromthe acoustic source 230 through rectified diffusion until itsubstantially fills the microcavity 904. The gas/liquid interface 914 ofthe fully formed bubble 908 displaces the liquid fluid immediatelyadjacent to the interface 914, imparting localized fluid movement. Thisprocess may be repeated as necessary to create localized fluid movement.

With further reference to FIG. 9, there is a relationship betweenfrequency of the acoustic field 240 and the diameter of the resonantbubble 914 in a free aqueous fluid; namely, frequency in Hertzmultiplied by resonant the bubble radius is approximately equal to threemeters/second. For example, if a transducer having a frequency of about1.1 MHz is focused, a resonant bubble of about radius of 2.7 micronsforms. Similarly, if a transducer having a frequency of about 11 MHz isfocussed, a bubble having a radius of about 0.27 micron forms. Therelationship becomes more complicated when the bubble is in orinfluenced by a microcavity or concave feature on a surface.Nevertheless, this dimension is appropriate for microfluidic andmicro-electro-mechanical-system (MEMS) type devices. It is an propertyof the invention that it is possible to utilize a 1.1 MHz focusedtransducer to control cavitation in a miniaturized device to regionshaving micron scale dimensions as the focal zone of this transducer isrelatively large at about 2×6 mm.

In a further illustrative embodiment, the mixing apparatus of theinvention can be employed to micromix fluid surrounding an activedetection or reaction site, such as a DNA spot in an array of DNA spots.FIG. 10 is a conceptual diagram 1000 depicting operation of an acousticmicrostreaming-based mixing apparatus according to an illustrativeembodiment of the invention. FIG. 10 shows a cross-section of amicrofluidic device 1002 with an active detector site 1004, such as aspot containing DNA probes or an electrode. The device 1002 alsoincludes nucleation promoting features 1006 a and 1006 b. Optionally,the site 1004 may be placed on an independent zone 1008 that isacoustically masked from the active acoustic field 240. The oscillationof the gas body or bubble 1010 in a fluid 1012 contained within amicrochamber formed by the two structures 1014 and 1016 provides a localmicromixing action around the feature 1006 a indicated by the arrows1018 a and 1018 b; and the oscillation of the bubble 1011 provides alocal micromixing action around the feature 1006 b indicated by thearrows 1020 a and 1020 b. Thus, the invention can be employed to micromix portions of the fluid 1012, without disturbing the active site 1004.Skilled artisans will appreciate that the acoustic source 230 and/or thecontroller 410 may be integrated with or remotely coupled to the device1002. By way of example, the source 230 may be located either inside thechamber formed by the elements 1014 and 1016, or integrally formed to anexternal wall of one of the elements 1014 and 1016. Alternatively, theacoustic source 230 can be located remotely as shown in FIG. 1.

FIG. 11 is a conceptual block diagram 1100 illustrating use of themixing apparatus to locally mix fluid in a microdevice containing anarray of active sites. More particularly, FIG. 11 depicts a fabricateddevice 1102 including a plurality of active detector sites 1104, witheach active site 1104 surrounded by a plurality of selectively placednucleation features 1106. Although not shown, skilled artisans willappreciate that in practice, the microarray 1102 employs a cover, suchas the element 1014 in FIG. 10, to form a microchamber containing thearray of active sites 1104 and nucleation features 1106. According tothe invention, in response to exciting the nucleation features 1006 withthe acoustic field 240, micromixing of the liquid occurs proximate toeach active site 1104, without dislodging the active componentscontained in each site 1104. Although FIG. 11 depicts a specificarrangement of nucleation features 1106 around each active site 1104,skilled artisans will appreciate that the nucleation features 1106 maybe located anywhere within the microchamber of device 1102 relative tothe detector sites 1104, and that such location can be selected toeffect fluid flow within the chamber. For example, if high fluidvelocities or pulsating bubbles cause damage to the active sites 1104 ina particular device 1102, the nucleation features 1106 can be located acertain minimum distance from the detector sites 1104. Alternatively,the active sites 1104 can be located away from the nucleation features1106. The relative positioning of the nucleation features 1106 and theactive sites 1104 can be optimized to enhance flow of the chamber fluidpast the active sites 1104 without scrubbing or otherwise damaging theactive sites 1104.

In a further embodiment, nucleation features may be located in theactive sites 1104 to effectuate mixing. Also, as described above, theacoustic source 230 may be located as depicted in FIGS. 1 and 7 relativeto the microarray 1102. Alternatively, the acoustic source 230 may beintegrally formed to the top (cover not shown) or bottom 1108 externalsurface of the array 1102, or contained within the array microchamber.

Thus, the invention may be utilized to accelerate synthesis and bindingreactions at the active sites 1104. The result is a controlled,micromixing apparatus capable of scaling the magnitude of mixing to theapplication required. Further, if the acoustic energy beam is bothfocussed and scanned the sequential activation of bubbles, such as thoseoccurring at the nucleation features 1106, can be employed to produce anet directional flow in the surrounding fluid.

With the acoustic source 230 and/or the controller 410 being able to befabricated integrally with or separately from the microarray 1102 orother microdevice, the invention can provide a compact testing device.One particular application is in the formation of a DNA testingcassette. In one embodiment, the mixing apparatus is incorporated into acassette holding a hybridization slide having a cover with fluid portsat either end. Connections are provided to fluidic reservoirs to washwith different solutions. The mixing apparatus can be orientedvertically to enable gravity-based fluid flow to be accelerated with thesonic energy mixing. The sonic energy field established can be used toprovide energy to disrupt surface tensions to allow fluid flow acrossthe array or other immobilized surfaces. Alternatively, the mixingapparatus can be oriented horizontally with hydrostatic pressuredifferentials across the reaction chamber to drive the fluid transfer.The surface tension of a low profile system (such as 100 micrometer gapheight) may be altered with the sonic energy field to allow faster andmore uniform slide processing.

One important aspect of the invention that enables the mixing apparatusto achieve the above discussed effects is the creation and selectiveplacement of nucleation features, such as the feature 804 and themicrocavity 904. Such features reliably induce (nucleate) bubbleformation and enable cavitation acoustic energies lower than thatrequired to cause cavitation on relatively smooth surfaces or insolution. Such nucleation features may be placed to control wherecavitation occurs. These nucleation features can be in the form ofdiscrete point-like features, including but not limited to pits,crevices, craters, frustums, pins, posts spikes, spicules, bumps orlinear features, including but not limited to scratches, grooves,ridges, or the like. The nucleation features can be produced by diversemanufacturing methods including but not limited to scratching, etching,grinding, engraving, milling, drilling, sand blasting, ion-beamprocessing, molding, pressing, hot stamping, microlithography,micromachining, microfabrication or the like. In addition to geometricfeatures, localized variations in material properties such as acousticimpedance, density, modulus of elasticity, hydrophobicity, wetability,surface energy, distribution of impurities or contaminants on or in asurface, and the like can also preferentially nucleate cavitation. Thesematerial variations may be formed by manufacturing processes includingbut not limited to ion implantation, plating, chemical modification, orthe like. Nucleation features can also be created by placement orformation of electrodes.

Geometric or material features may also be located on the outside of adevice to promote cavitation on the inside of the microdevice byinteracting with the acoustic field to cause intensity variations in theacoustic field coupled into the microdevice.

When the density of features is high, as for example in the holes of amicroporous membrane, or a nonspecific etching or grinding leading to afrosted effect, then the multitude of features may be called a“texture”. The textures may be either ordered as in an array offeatures, as discussed above with respect to FIG. 11, or includerandomly distributed features. The extent of textured areas may becontrolled so as to promote cavitation in specific regions of amicrodevice, such as discussed above with respect to the regionsurrounding each active site 1104 of FIG. 11. As discussed in furtherdetail below, the creation of features and textures enables thegeneration, of zones of mixing and pumping which can be selectivelyactivated by acoustic energy. As discussed above, the selectiveplacement of the cavitation features enables local micromixing of afluid at selective locations while using an unfocused acoustic source.Thus, enabling an acoustic source to be mounted directly to microdevice.

Bubbles may also be generated by other means, such as chemical reactionor by electrochemical processes. For example, bubble generation sites inthe microdevice may have or contain an immobilized chemical species thatwill react with a species in the fluid either when brought into contactor in response to a change in condition such as a change in pH. Anothermeans of generating gas bubbles is by electrolysis at an electrode.Applying an appropriate electrical signal to an electrode in contactwith an appropriate fluid in a microdevice will cause gas bubbles toform. Other electrochemical means may generate other gas species usefulfor the method of this invention. Electrochemical means of gasgeneration in the fluid of the microdevice can be controlled bymodulating the electrical signal applied to the electrode. Gas bubblesmay be generated in regions in a device by arrays of chemically orelectrochemically active features or by making an entire regionchemically or electrochemically active. The bubbles that are generatedchemically or electrochemically may caused to grow by further gasproduction by chemical or electrochemical reaction or in response to anacoustic field. The bubbles may then release and stream in response toan applied acoustic field. The streaming bubbles cause fluid flowthrough viscous and momentum effects.

The size of the features or extent of a textured area may be smaller,even much smaller, than the focal zone of the acoustic field 240, if itis focused, or the extent of an unfocused field. This enables control ofcavitation to a much smaller scale than a wavelength of the acousticfield 240.

According to one embodiment, the nucleation features are located tocreate rotational flow features that resemble the eddies that occur inturbulent flow. In another embodiment, the acoustic source 230 is directat naturally occurring nucleation features to create rotational flow.Such an approach adds vorticity to what would otherwise be totallyirrotational flow. In one embodiment, eddies are created near the wallsor margins of conduits or cavities, which are the regions that are themost difficult to mix with alternative technologies. These eddiesgreatly enhance fluid exchange and mass transfer within the conduits orcavities of a small fluid microdevice. The distribution of eddy sizesand loci within a microdevice can be tailored to resemble those of aturbulent flow field. The distribution of sizes and loci can beoptimized for particular microdevices and applications such as flushinga microchamber or mixing reactants during a heterogeneous hybridizationreaction.

FIGS. 12A and 12B depict the use of the apparatus to provide flowcontrol in a microconduit. More particularly, in FIG. 11A, the elements1202 and 1204 are located to form an exemplary microconduit 1206.According to an illustrative embodiment of the invention, the element1204 includes a nucleation feature 1208. The nucleation feature 1208 maybe naturally occurring, such as being the result of fabrication, orparticularly created. In response to the nucleation feature 1208 beingsubmerged in a fluid, a gas pocket 1210 forms in the feature 1208. Asshown in FIG. 12B, when the nucleation feature 1208 aligns with theacoustic source 230 and the acoustic field 240 applies pressure, thepocket 1210 forms into a bubble 1212. The bubble 1212 effectively stopsfluid flow, indicated by the arrow 1214. Upon cessation of the appliedacoustic field 230, the bubble 1212 dissipates enabling fluid once againto flow through the conduit 1206. The bubble 1212 functions essentiallyas a microflow control valve for conduit 1214. As discussed above, theacoustic field may be unfocused or particularly directed at thenucleation feature 1212.

In the case of flowing fluid in laminar flow conditions, the fluid nearthe walls of a conduit, such as the conduit 1214, or a chamber, is notmixed into the core of the flow, in the center of the conduit or cavity.If the fluid in the conduit or chamber is to be replaced by anotherfluid, as in a flushing application, adding vorticity with acousticmixing can dramatically reduce the time and fluid volume required toachieve a required dilution.

In the case of a device with no flow, such as a reaction chamber duringincubation, acoustic mixing can effect mass transfer that would onlyoccur otherwise as a result of diffusion. The invention is especiallyuseful in the case of reactions involving large biomolecules, wherediffusion rates are extremely low. These reactions are rate limited bythe diffusion of the reactants. Heterogeneous reactions involving areactant that is bound to the surface of the reaction chamber, can begreatly accelerated using the methodology of the invention.

FIG. 13 is a conceptual diagram 1300 depicting the use of the acousticmixing apparatus to cause fluid flow in a microdevice according to anillustrative embodiment of the invention. In a similar fashion to thedevice of FIG. 12, the elements 1302 and 1304 are located to create amicroconduit 1306 though which fluid can flow (indicated by the arrows1310). The element 1304 includes a nucleation feature 1308 for promotingnucleation. Once again, the nucleation features 1308 can be naturallyoccurring or particularly created and placed. In the presence of anappropriate acoustic field 240, feature 1308 will generate and releasecavitation bubbles. The released bubbles will stream in response toacoustic field intensity gradients. Viscous and inertial effects willcause fluid motion in the same direction. The direction and rate of flowis controlled by the intensity and local intensity gradient of theacoustic field 240. Acoustic field gradients may result from focusingthe acoustic source 230 or be established by the deployment ofabsorptive or reflective materials, either between the acoustic source230 and the fluid volume, or on the far side of the fluid volumerelative to the acoustic source 230, so as to influence either incidentor reflected acoustic radiation. Thus, the embodiment of FIG. 13provides controlled, directional pumping of bulk fluid.

FIGS. 14A-14D are conceptual diagrams depicting an alternativeembodiment of the invention for providing fluid pumping. FIGS. 14A-14Ddepict progressive states of a microdevice 1402. The microdevice 1402includes a series of acoustic transducers 1404 a-1404 c, a chamber 1406containing fluid having the moieties 1408, and textures 1410 formed in asurface 1412. As in the previous illustrations, the nucleation features1410 can be naturally occurring during fabrication or particularlyformed and positioned. Additionally, the acoustic transducers 1404a-1404 c can be integrally fabricated with the microdevice 1402 or canbe remotely located. Also, the acoustic transducers 1404 a-1404 c can beseparate, individual transducers, or alternatively, a single remotelylocated source can be directed at any one or all of the regions 1416a-1416 c of the microdevice 1402.

To simplify the illustration, the controller 410 is not shown, but maybe included in the device 1402 or may be communicatively coupled to theacoustic transducers 1404 a-1404 c. As shown in FIG. 14B, by activatingthe acoustic transducer 1404 a to form bubble(s) 1414 according to themethods described above, the moieties 1408 can be moved from zone 1416 ato 1416 b. As shown in FIG. 14C, by activating the transducers 1404 aand 1404 b, in sequence, the bubble 1414 can be formed to flow themoieties 1408 into zone 1416 c. Similarly, by activating the transducers1404 a, 1404 b and 1404 c in sequence, the bubble 1414 can be formed toflow the moieties 1408 to the far edge of zone 1416 c.

Skilled artisans will appreciate that the moieties 1408 may be processedor analyzed differently in each of the zones 1416 a-1416 c. For example,a biological tissue sample, such as the sample 1408, may be insertedinto a device, such as the device 1402 in a particular zone, such as thezone 1416 a. The tissue 1408 can be introduced to a disruptive acousticenergy field from the transducer 1404 a and the disrupted tissue 1408 isthen ready for reagent addition. The reagent addition may occur in zone1416 b, which has acoustic mixing conditions (e.g., to accelerate enzymereactions). The reaction products may then be transferred to zone 1416 cand the binding (e.g., hybridization) events may be improved withacoustic energy. One integrated device 1402 may have multiple zones fordifferent processes, so that all of the above processing can occur on asingle device. As mentioned above, the microdevice 1402 may, forexample, be inserted into an external acoustic field, or processing canbe accomplished by on-board acoustic field generation. This isespecially appropriate for very small sample masses, for example, in themicrogram to picogram range.

Although the above described illustrative embodiments contemplate achamber, such as depicted in FIG. 10 at 1012, for enclosing a liquid,the illustrative acoustic mixing apparatus can also be employed forlocalized mixing in a small volume of liquid on a surface of in a wellas shown in FIG. 7, without a fully enclosed chamber such as depicted inFIG. 10. By way of example, FIG. 15 is a conceptual diagram 1500depicting the use of the invention for mixing a solution on a microscopeslide. More specifically, the diagram 1500 shows a microscope slide 1502having a nucleation site 1504. The liquid to be mixed 1506 is placed onthe slide 1502. As a result, a small gas/vapor body 1508 forms with agas/fluid interface 1510. Upon application of the acoustic field 240,the body 1508 expands to become the bubble 1512. As described above withrespect to FIG. 8, the cyclic nature of the acoustic field 240 causesthe bubble 1512 to cavitate. Alternatively applying and removing theacoustic filed 230 causes the bubble 912 to oscillate, thus providing acyclic displacement of fluid adjacent to the interface 1510, and a localmicromixing action in the fluid 1506 on the slide 1502.

One advantages of the mixing technology of the invention is that bylocating or making use of cavitation features close to the edges ormargins of the chamber of conduit, with the invention the highest fluidflow velocities are achieved at those locations. Other methods whichprovide bulk fluid flow cause the highest velocities to occur near thecenter of the chamber or conduit where they are less useful forapplications such as flushing or reacting solution phase molecules withthose bound to a surface. Another advantage of the methodology of theinvention is that mixing can be achieved in a rigid microdevice. Othertechnologies require that the chamber or conduit to be mixed have aflexible member (such as a plastic film contacting a liquid fluid or anaqueous/organic liquid fluid interface) that can be deflected byexternal means to cause bulk fluid motion within the device. No suchflexible member is required with the invention. A further advantage ofthe invention is that nucleation sites or textures can be built into amicrodevice such that mixing or fluid flow occurs only in desiredlocations. This is useful for controlling the extent and location ofhigh velocity flows that may have a detrimental effect on sensitiveareas within the device.

Another advantage of the invention is that it can operate either as anintegrated component of a microdevice or as a separately fabricated, butacoustically coupled add-on. In a further embodiment of the invention,the localized bubble formation and collapse created by the interactionof the acoustic field 240 with nucleation features can be employed toclean electrodes in a microdevice. One of the difficulties of performingelectrophoresis in a miniaturized format is the scaling effects ofminiaturization. Another difficulty is the presence of bubbles.Bubble-effects can block internal lumen, thereby rendering a sample/chipinvalid. Another limitation is the intrinsic properties of theelectrophoresis process; namely, the generation of bubbles from theelectrolysis of water at noble metal electrodes. This bubble formationmay have detrimental effects in miniaturized formats by blocking fluidflow, and by affecting the efficiency of the electrophoresis by limitingthe current. Cleaning the electrodes can minimize some of thesedifficulties. The invention may also be used to sweep across theelectrophoresis zone to mechanically agitate the molecules to beseparated.

FIGS. 16A and 16B are conceptual block diagrams depicting amicro-electrode cleaning device according to an illustrative embodimentof the invention. The microdevice 1602 is composed of two elements 1004and 1006 arranged to form a chamber or conduit 1608. The element 1604includes a nucleation promoting feature 1610. An electrode 1612 ismounted on the element 1606 and includes an electrically conductive lead1614. The chamber 1608 contains a liquid fluid. By causing a bubble 1616to form and collapse using the methods described above, the mixingapparatus of the invention can be employed to clean the electrode 1012.

FIG. 17 depicts another embodiment in which bubble nucleation isfacilitated by electrolysis at an electrode. An electrical current isapplied via a connection 1706 to an electrode 1704 mounted on asubstrate 1702 and in contact with a fluid medium 1708 such thathydrogen (or other gas) bubble(s) is generated. Growth of the bubble maybe either by continued generation of gas by electrolysis or bysubsequent interaction with the acoustic field 240. In the presence ofan appropriate acoustic field 230, the bubble(s) will grow, release fromthe nucleation site and stream in response to gradients in the acousticfield. The size of the electrolysis bubbles and the frequency of theiroccurrence can be controlled by the electrical signal to the electrode1704. Many bubbles may be generated on an electrode 1704 or manyindividual electrodes 1704 or conductive sites may be employed.Nucleation may be controlled by means of electrical signals applied tothe sites rather than by an acoustical source. This method may be usedto cause controlled nucleation of cavitation events at specificlocations or in specific regions at specific times without themicrodevice being movable relative to the acoustic source 230. Forexample, a device may have an integrated non-focused acoustic source230. Bubble streaming and fluid flow can be activated in specificregions of the microdevice as required by controlled activation ofelectrodes 1704 in the regions. Acoustic field gradients may bepre-established by the deployment of absorptive or reflective materials,either between the acoustic source 230 and the fluid volume 1708, or onthe far side of the fluid volume 1708, relative to the acoustic source230, so as to influence either incident or reflected acoustic radiation.

FIG. 18 is a conceptual diagram depicting an acoustic based mixingapparatus that incorporates the acoustic source into a microfluidicdevice according to an illustrative embodiment of the invention. FIG. 18shows a cross-section of a microfluidic device 1800 with an acousticsource 1890 which is connected to an electronic controller (not shown)by connections 1870 and 1880. The device 1800 also includes nucleationpromoting features 1850 which may be located in the microdevice oppositethe acoustic source as shown or on the surface of the source itself. Asshown in the figure, the acoustic energy drives oscillation of a gasbody or bubble 1810 in a fluid 1812 contained within a microchamber ormicroconduit formed by the two structures 1840 and 1845 provides a localmicromixing action around the features 1850 indicated by the arrows1820. The acoustic source can also cause fluid motion by streaming ofbubbles as shown in FIG. 13.

The acoustic source 1890 can form an entire internal surface of themicrodevice, can form a portion of an internal surface of themicrodevice or can be attached to an internal surface of the device. Theacoustic source 1890 can be integrally fabricated with the microdeviceor may be separable from the microdevice so as to be reusable. A thinfilm or membrane 1846 may separate the integrated acoustic source 1890from the fluid 1812. The membrane 1846 may be tailored to facilitate orblock the transmission of the acoustic energy from the acoustic source1890 into the fluid 1812 in selected areas.

The acoustic source 1890 may be any material or structure that emitsacoustic energy such as but not limited to piezoelectric elements,magnetorestrictive elements, capacitive micromachined ultrasonictransducer (cMUT) elements and the like. The acoustic source may befabricated in situ as part of the microdevice by any manufacturingprocess such as but not limited to additive and subtractive processesperformed in micro-electro mechanical systems (MEMS) fabrication or maybe fabricated as a separate device and integrated into the microdevicein a subsequent manufacturing operation.

The acoustic waves generated by the source 230 can be any of a largevariety of acoustic wavetrains characterized by being able to generatecavitation bubbles at nucleation sites of structural or materialfeatures at a power density below that needed to generate bubbles innon-featured regions. An acoustic source can be unfocussed, or can havea line (one-dimensional focus), or can be focussed in two dimensions toa spot. In line and spot focus, there is also focus in the thirddimension, which is usually controlled by the focus in the otherdimensions.

Each of these focal modes is useful in particular embodiments of theinvention. Spot focus is particularly useful for focussing on particularwells or spots in an array, such as the array 1102 of active sites 1104of FIG. 11. Line focus can be used effectively to sweep an acousticfield across an array, thereby either simultaneously treating wells orspots, such as the detector sites 1104, by the row, or driving fluidthrough an array in a particular direction. Non-focus (planar) waves areparticularly effective for causing stirring or heating in the entirearray (e.g., FIG. 11), plate (e.g., FIG. 15), or other object oftreatment. In some protocols, one of these modes is sufficient. In otherapplications, two or more may be needed. In multiple protocolapplications, it may be most cost-effective to provide independentsources of plane, line and spot focus.

In another illustrative embodiment, the mixing apparatus has two sonicenergy transducers/sources focused on two walls of a fluid containmentvessel/microdevice. The reagent chamber, such as the chamber 1012 ofFIG. 10, volume is narrow to contain the fluid when the transducers arenot activated. When one or more of the transducers is activated thefluid is pumped out of the reagent chamber. The transducers may beoriented such that planar standing waves form. In addition, thetransducers may be synchronized such that the deflection of the wallsresults in a maximum narrowing of the lumen or a maximal expansion ofthe lumen.

In another illustrative embodiment, the mixing apparatus employs anunfocused acoustic energy source. For example, the field of textures maybe very close to or a part of the transducer, such as a MEMS fabricatedtransducer with on-board nucleation site(s).

In another illustrative embodiment, the energy input rate from theacoustic source is used to modulate the temperature of the fluid. Thisbenefits hybridization-based processes, and potentially other bindingprocesses. Since the acoustic energy is able to provide mixing of thefluidic solutions surrounding solid-phase, immobilized binding partners,mismatches may be minimized. Adjusting the temperature during theprocess can also improve binding characteristics.

In a further illustrative embodiment, the mixing apparatus is used tomix a fluid in a miniaturized chamber. One embodiment consists of athick physical layer, a fluidic zone containing sample to be analyzed,and a thin physical layer. Materials that are appropriate for thephysical layers include glass, polymers, and plastics. The focusedacoustic energy is directed to cavitate in the free fluid with theresultant shock wave impinging on the exterior of the device. Theimpedance mismatches between the layers result in resonance of one ofthe layers. This compression-rarefaction vibration process inducesmixing within the chamber. In a sense, the acoustic impedance mismatchdrives an oscillating diaphragm.

In another illustrative embodiment, the mixing apparatus is used todegas solutions. This may be useful for controlling dissolved gasses ina sample or reagent that is being loaded or has been loaded in a device.In this application, the sample is loaded into a region of themicrodevice that is in the focal point of the converged acoustic beam.Insonifying the region will prompt dissolved gas to be released prior tothe sample entering the microfluidics.

In another illustrative embodiment, the mixing apparatus of theinvention is employed to facilitate or cause the aggregation andmanipulation of small particles in small devices such as hybridizationchambers that are used with DNA chips. In this embodiment, the mixingapparatus includes a source 230 for generating a focused acoustic field240 that is arranged perpendicular to the plane of a hybridizationchamber with a coupling medium (e.g., water) interposed. The focal zoneof the acoustic field 230 is positioned near or at the plane of thehybridization chamber. The chamber can be moved relative to the acousticfield in the lateral or axial direction during or between treatments.The mixing apparatus is configured such that the transducer faces upwardin a water bath and the hybridization chamber is positioned horizontallyat or near the surface of the water bath. The air interface at the topside of the hybridization chamber forms an acoustically reflectivesurface that creates a standing-wave acoustic field within themicrodevice. The reflector may also be a metal plate or any othermaterial that effectively reflects sound. At the scale of these devices,an ultrasound transducer operating at 1.1 MHz produces a focal zonehaving an appropriate size and a gaussian intensity profile in the focalplane. Other frequencies may be used and other intensity profiles may beappropriate, such as a monotonic intensity gradient produced by anunfocused transducer operating at an oblique angle to the plane of thehybridization chamber or fluidic device.

In illustrative experiments directed to mixing in a hybridizationchamber, particles in suspensions such as milk or ink have been observedaggregating in patterns around the focal zone in the chamber. Thishappens in low intensity fields, typically under about 100 mV (appliedto amplifier). If the chamber is moved after the particles areaggregated, the pattern will change rapidly to find the energy minimalin the newly formed standing-wave field.

In another embodiment, the invention employs mixed frequencies to affectan acoustic field. For example, a brief tone burst at a high intensityat a high frequency to form acoustic bubbles may be followed by a toneburst at a lower frequency to retain the bubble(s) bolus and also softenthe bubble collapse. In addition, varying the frequency will also varythe focal zone location which may be beneficial for certainapplications.

In another embodiment of the invention, the acoustic 230 source has acylindrical segment focused inward, such as a 45 degree arc of acylinder to result in a line focal zone, e.g., about 1.5 mm wide×about 4mm long×about 50 mm deep. If a microwell plate was swept across thefocal zone line, an entire plate of small sample volumes such as the1,536 wells of 1 microliter, may be rapidly treated.

A further application of the invention relates to histochemistry.Histochemistry is the staining of tissue, particularly tissue sectionsand biopsies, for detection of diseases and other pathological states.It typically involves the sequential addition of numerous reagents tosmall samples for particular times, followed by washing out of thereagent and addition of a second reagent. In addition to staining,tissues may be dried, embedded, demineralized, and otherwise processed.Modem methods may involve specific labeling with protein or nucleic-acidbased reagents.

The ability of the devices of the invention to provide gentle localstirring means that diffusional mixing in the liquid is no longer alimiting factor in the rate of processing. In some procedures, thiscould significantly decrease processing time. Moreover, the techniquesof the invention allow remote control of fluid flow, which couldsimplify and help automate the techniques.

Principles of the invention can be further understood through thefollowing illustrative experimental examples.

EXAMPLE ONE Cavitation Promoting Sites

A glass slide was scratched with a diamond scribe. The slide was placedin a slide holder and installed scratch-down in the apparatus of FIG. 1,modified by the addition of a 5 MHz cavitation detection transducerplaced confocally with the main 1.1 MHz acoustic power transducer 230.This instrument was used for all the following examples except wherenoted. A process was configured to sweep the slide through the focalpoint of the convergent acoustic beam of the power transducer. The glassslide was insonified with a treatment waveform generated by a 100 mV, 1%duty cycle, 1000 cycles per burst signal applied to a 55 dB RF amplifierand input to the power transducer. Both the input signal to the RFamplifier and the output signal from the cavitation detection transducerwere applied to channels 1 and 2 of a Tektronix TDS 30334 digitizingoscilloscope. The signal from the cavitation detection transducer wasprocessed in real time to create a FFT frequency spectrum which wasdisplayed on the oscilloscope screen along with the time-domain signals

There was no signal above the noise floor from the cavitation detectiontransducer as the undamaged surface of the glass slide moved through thefocal zone. As the scratch crossed through the focal zone, a significantsteady signal from the cavitation detection transducer was recorded bythe oscilloscope. This resulted from cavitation in the vicinity of thescratch on the glass slide. Repeating this experiment with an inputamplitude of 150 mV caused sporadic large amplitude signals to begenerated by the cavitation detection transducer whether or not thescratch was in the focal zone.

Example 1 illustrates that the output signal from the cavitationdetection transducer can be processed and one or more characteristics ofthe signal can be employed in a feedback control mechanism to controlthe intensity and nature (stable vs. transient) of the cavitation.Example 1 also illustrates that stable and transient cavitation exhibitsignificantly distinct acoustic signatures that can be distinguished anddifferentiated by electronic or computer processing, and that surfacefeatures or textures promote cavitation.

EXAMPLE TWO Mixing With Particles in a Acoustic Field

We conducted a number of experiments with dyes and water in a chamber ona microscope slide. The chamber was 12 mm in diameter and 0.5 mm high.We applied dye and water in the chamber and placed it in a sampleholder, such as those described above with respect to FIGS. 1-7. Weadjusted the water level in the tank such that the top of the slide wasnot covered by water. This creates a standing-wave sound field wheninsonified from below. We insonified the chamber with a wide range oftreatments. These treatment caused noticeable but not dramatic mixing.We then added glass microspheres of the type used to thicken epoxy tothe chamber. In the standing-wave acoustic field, the microspherestranslated and aggregated at the acoustic nodes. By dithering thechamber in two dimensions in the acoustic field, the particles wereforced to translate within the chamber and thus, mix the fluid in thechamber. We noticed a significant increase in mixing efficiency with theaddition of the particles. This example illustrates that by addingparticles to the fluid in a chamber and by dithering the chamberrelative to a standing-wave sound field, mixing is enhanced.

EXAMPLE THREE Selectively Blocking an Acoustic Field

If a microdevice or region to be treated is smaller than the focalregion of a convergent acoustic beam, or if it is necessary to protectportions of the microdevice from the sound field, acoustic blockingmaterials may be placed to shadow selected areas from the acousticsource. To illustrate this and its application to MEMS devices, anacoustic blocking material (Tyvek) was applied to the outside of achamber containing a piece of foil. The result was that cavitationdamage preferentially occurs on the unblocked portion of the foil.

EXAMPLE FOUR Cavitating Inside of a Chamber Without Cavitating on theOutside of the Device

An aluminum foil strip was placed in a Grace Bio-Labs #PC20 chamberfilled with water and clamped to a microscope slide. The chamber wasthen placed, cover side down, in a bath of degassed distilled water inthe apparatus of FIG. 1. The surface of the glass slide was positionedperpendicular to the focal axis and at a distance of 2.4 mm from thefocal plane towards the transducer. The chamber was insonified with awaveform of 300 mV, 10% duty cycle and 1,000 cycles per burst.Cavitation damage (pitting) occurred on the foil within the focal zoneof the acoustic field. No damage was observed on the outside of thechamber. This illustrates the ability to control cavitation by means ofmaterials, geometry and wavetrain parameters to cause cavitation tooccur at a specific location within a device.

EXAMPLE FIVE Improved Hybridization From Mixing During Incubation of aDNA Microarray

A chamber was constructed using a standard microscope slide for a base,a 1 mm thick by 25 mm square glass cover and laminated spacers along twoedges. The spacers each included a 10 micron thick Osmonics Poreticsmembrane (5 micron pore size) and a 37 micron polyester shim with theedges aligned inside the chamber, resulting in a 47 micron gap betweenthe slide and the cover. The chamber was clamped with small steel binderclips over each of the two spacers. The other two edges were left open.Acoustic energy was applied to the edge of the membrane/shim laminate bymeans of the apparatus of FIG. 1. A focused ultrasonic transducer (SonicConcepts #H101) operating at a frequency of 1.1 MHz. and directed upwardalong an axis perpendicular to the plane of the glass slide. The slidewas positioned horizontally at the surface of a water bath, coincidentwith the focal zone of the transducer. The slide was held fixed in thevertical direction but movable in all horizontal directions. Theposition of the slide is typically dithered in the horizontal planeduring treatment to minimize the effects of misalignment of the sliderelative to the transducer and to expose as many nucleation sites aspossible along the edge of the membrane to the focal zone of theacoustic field.

Mixing Ink Particles in an Open Chamber (Cover Slip).

White ink was refined by serial centrifugation to obtain sub-micron TiO2particles. These were resuspended in 1×SSC. The ink particle solutionwas applied to one edge of the chamber and allowed to wick into the gapand fill the chamber. Several acoustic waveforms were tested. Thefollowing waveforms of Table 1 were found to cause the fluid to jet fromthe edge into the center of the chamber, as determined by visualobservation.

TABLE 1 Voltage (mV)* Duty cycle (%) Cycles per burst 70/40 100 10/500030 100 (cw) 2000 70 10  10 70/30 50 10/2500 *The voltage is the inputvoltage to a 55 dB RF amplifier. The output of the amplifier was appliedto the transducer through an impedance matching network.

The effective mixing distance from the edge of the chamber was forced tocorrelate with the acoustic power applied to the slide. Higher powerwaveforms caused ink particles to move from one side of the chamber tothe other, a distance of approximately 22 mm.

Accelerating DNA Hybridization in an Open Chamber (Cover Slip).

The chamber described above was applied to a glass slide on the surfaceof which was spotted a DNA array of gene probes. A target solution ofcDNA in a hybridization buffer was applied to the chamber and incubatedfor 2 hours at 65° C. while being treated with an acoustic field topromote mixing in the chamber. A similar array was subjected to the sameconditions without the acoustic treatment. The treatment consisted ofexposure to an acoustic waveform of 70 mV, 10% duty cycle and 10 cyclesper burst for approximately 5 minutes at 30 minute intervals. Aftertreatment the two slides were post-processed together.

The two slides were scanned with a GSI Lumonics scanner. The imagesproduced show that the treated slide had substantially more fluorescentsignal than the slide that was not acoustically treated.

EXAMPLE SIX Non-Contact Mixing in a Microliter Drop

A one microliter drop of milk and India ink was applied to the drysurface of a glass slide at the water/air interface and in the focalzone of a 1.1 MHz focused transducer. When 0.5 microliter of milk wasadded to 0.5 microliter of India ink, the solutions slowly mixed, butwhen an acoustic wavetrain was applied to the drop, the two solutionsrapidly mixed in less than 2 seconds. The energy applied from thetransducer to the drop had a peak positive pressure of approximately 3MPa at 1% duty cycle with 1,000 cycles per burst.

The experiment was repeated using a glass slide which had been scratchedwith a diamond scribe to create a crevice approximately 25 microns inwidth. The crevice or pit acted as a nucleation site for bubbleformation. When the drop of fluid was located over the crevice, theacoustic intensity required to mix the fluids was reduced and the siteof nucleation was predicted. This illustrates the use of geometricfeatures to control the location of cavitation and reduce the acousticintensity required for cavitation and mixing.

EXAMPLE SEVEN Complex Wavetrain Mixing—Hammerhead Wavetrain

An experiment was performed using a complex wavetrain to control bubbleformation and collapse. A wavetrain was designed to have a short burstto nucleate and form a bubble or bubble cloud, followed by a loweramplitude period to allow the bubble(s) to slowly collapse, and followedby a period of no acoustic signal. The following conditions worked wellto both provide visual mixing in a dye filled chamber system and mixwithout disruption of immobilized DNA on polyLysine coated glass slides(i.e., following fluorescent dye staining), using the apparatus ofFIG. 1. At 1.1 MHz frequency, 10 cycles of 70 mV (amplifier input),followed by 2,500 cycles of 30 mV, and followed by 2,500 cycles of 0.1mV. This wavetrain was repeated and dithered across a field of scratchesin a glass microscope cover slide with 0.5 mm spaces. The ditheringparameters were a 2 rpm rotational velocity and a 2 mm radius with a 0second dwell time. This wavetrain was scanned across the textured field.The use of submicron particles of dye allowed visualization ofcontrolled mixing. The observation was a smoother and less sporadicmixing than that observed without the step-down from the high intensitycycles.

Experiments 8-14 were performed to understand the effect of differenttextures on mixing. The effect of texturing the top vs. the bottomsurface in the field and the effect of material of the textured surfacewere also determined. These experiments were performed using theapparatus of FIG. 1. The experiments were performed using a GraceBio-labs Coverwell PC20 20 microliter hybridization chamber clamped to astandard glass microscope slide. This chamber is 13 mm in diameter andforms a gap of about 125 micrometer when clamped to a flat surface. Wetested this configuration with the following conditions: Treatment: 10%duty cycle, 10 mV amplitude, 1,000 cycles per burstZ-offset equal to −9to −3 mm from the focal plane

EXAMPLE EIGHT

A chamber with no features (or textures) containing water and milk wasinsonified . Milk solids formed into a white spot at the center of thefocal zone, surrounded by a clear ring. Milk solids were aggregated andsegregated at the center of the focal zone. Dithering caused the whitespot to smear out and remix.

The experiment was repeated with a single scratch feature inside thecover well. The passive cavitation system connected to the oscilloscopeshowed a frequency spectrum containing 1.1 MHz peak and harmonics. Whenthe dither function passed the focal zone through the scratch, abroadband rise in the noise floor is produced corresponding to the passband of the receiving transducer (centered at 5 MHz ). At the same time,the milk was mixed rapidly and thoroughly into the water. It appearedthat the broadband cavitation signal correlated well with mixing in thechamber. The scratch was reoriented 90 degrees to rule out reflectionsas source of signal. The result was the same.

EXAMPLE NINE

A hatch pattern was scratched into a glass slide with a diamond scribeand assembled as above. The result was that the milk solids did not forma spot. This seemed to be because either the scratches broke up thestanding-wave field or the milk was being mixed faster than it couldsegregate.

EXAMPLE TEN

The previous experiment was repeated with ink added to the chamber tovisualize mixing. A 1 microliter drop of black ink (fountain pen ink)wasadded to the inside surface of the hybridization chamber and one or twodrops of whole milk were placed on the glass slide. When the two werebrought into contact and clamped, the excess milk and some of the inksqueezed out, leaving the chamber filled with milk and a distinct smearof ink.

The same parts as above (hatch pattern scratched into slide, unmodifiedcover well) were used to repeat the experiment with ink added. Results:A chamber with no scratches in the glass mixed slowly. The mainmechanism seemed to be the dither function dragging an aggregate of milksolids around the chamber. The chamber with the scratches in the glassslide mixed very rapidly; the chamber was mostly mixed within the first5 seconds. It was completely mixed after 5 seconds of no dither and 5seconds of dither. As a control, An identical configuration with milkand ink was assembled and put aside. After 2 hours, diffusion wasevident but the ink and milk were essentially unmixed.

EXAMPLE ELEVEN

Several variations of the features on the glass slide were tested: Heavyscratches vs. light scratches in hatch pattern, Parallel scratches,Pitted surface, Single pit in surface at the focal point. All of thesefeatures caused mixing to some degree, compared to an unmodified slide.The mixing took the form of 1, 2 or 3 large eddies or vortices whichformed lobes around the focal point, with the shape of clover leaves.Occasionally, the number of lobes would change as if transitioning fromone stable configuration to another.

EXAMPLE TWELVE

The following features were tested on the inside surface of the chamber:smooth scratches created by knife in a hatch pattern, rough scratchescreated by a burr in an electric tool. These cases were examined withthe cover well facing down, towards the transducer. In both cases, therewas no effect at 100 mV but good effect at 150 mV. For an unmodifiedcover well, there was no mixing effect at 150 mV. When the device wasturned over, with the glass slide facing the transducer, there was noeffect until the voltage was increased to 200 mV. Then, there was amoderate effect.

EXAMPLE THIRTEEN

A “floating” cover slip was tested and compared to a gasketed chamber.This configuration seems to be more popular in the field of DNAmicroarray research. A standard glass slide was cut down to 2″ long foruse as a cover slip. A hatch pattern was scratched into the slide with ascratch interval of about 1 mm. The slide was used as a cover slipbecause regular cover slips are too thin to scratch a pattern into. (Apattern could be etched into a standard cover slip, however). A slidewas set up in the slide holder and positioned in a Covaris E1 systemwith a −4 mm z offset. A 1 microliter drop of white ink (Rotring#597118) was put onto the slide and the cover slip was applied, hatchside down. Distilled water was wicked in to fill the gap between theslides. This caused a streak of ink between the slides. A process wasconfigured using 40 mV CW as the treatment. This caused excellent mixingin the chamber, especially when dithering was turned on.

EXAMPLE FOURTEEN

It is possible to have cavitation promoting nucleation sites and texturedetails on the exterior of sample chamber to promote thebubble-formation and resultant collapse over the areas to be mixed onthe interior. The previous experiment was repeated with the cover slipturned upside down. This also was effective at mixing, although not asgood as with the scratches on the inside. An identical cover slipwithout scratches was tested. This did not mix. Particles aggregated ina bulls eye pattern at the focus. This may be beneficial for arrays thatare noncovalently attached to an interior surface.

EXAMPLE FIFTEEN Field Mixing With Membrane Bonded to Cover Slip

An Osmonics polycarbonate membrane with 5 micrometer pores was bonded toa 1 mm thick glass cover slip. The gap between the slide and the coverslip was set at 50 micrometers with plastic shims. White dye particlesin water were introduced into the gap. When the device was insonifiedwith an acoustic waveform of 50 mV amplitude, 10% duty cycle and 10cycles per burst, good fluid flow was observed in the plane of thedevice.

EXAMPLE SIXTEEN Edge Mixing

An array field may be constructed such that there are zones fornucleation and bubble collapse that act as pumps to stream fluid acrossthe array. If the energy is high the resulting bubble collapse maydisrupt bound binding partners. This may be a benefit to indicate inpost-mixing scanning of the array that efficient mixing occurred. Inaddition, the inclusion of various areas to indicate other aspects ofthe efficiency of the mixing may also be incorporated into the array.For example, areas that should have uniform amounts of dye may bedispersed across the array to indicate both mixing occurrence andefficiency.

An experiment was conducted to test edge mixing. An Osmonics membranewas sandwiched with plastic shim material with a glass microscope slidecover slip. The construction was (from the acoustic source) DNA arraymicroscope slide, 0.0375 mm plastic film, 0.010 mm membrane (OsmonicsPoretics PCTE, 5 micron pore size) and the cover slip (a 25 mm×40 mmborosilicate glass microscope slide). When the acoustic focal zone wasapplied to the fluid/membrane-shim interface the membrane acted as asource of nucleation such that the energy required to initiate bubbleformation was lowered. For example, 10 cycles of 70 mV followed by 5,000cycles of 40 mV readily mixed a fluid solution. The fluid flow was inthe plane of the array and perpendicular to and originating from themembrane edges. The flow was steady and was approximately 1 mm persecond. The flow pattern was from a point of approximately 1 mm andflowed over 10 mm perpendicular to the wall where the flow fanned outslightly to 3 mm. Adjusting the incoming voltages adjusted the velocity.

EXAMPLE SEVENTEEN Shelf or Ledge Mixing

In a hybrid configuration, a ledge or shelf of exposed membrane materialcan be constructed at the edge of an array. This mixes in a mode similarto the above described field mixing but is positioned at the edge of thearray. It has the advantage of creating a small gap in the mixing area(which requires less energy to activate) while allowing a larger gapover the array. A 5 micron pore size Osmonics membrane was bonded to ashim such that a portion of the face of the membrane was exposed withinthe device. When treated with a “pulse-step” waveform having 10 cyclesat 70 mV, 5000 cycles at 40 mV and no dead time, moderate mixing wasobserved within the chamber.

EXAMPLE EIGHTEEN Acoustic-based Temperature Cycling

The temperature of a hybridization chamber may also be modulated by therate at which the acoustic wavetrain enters the sample. A glassmicroscope slide with a hybridization chamber was oriented horizontallya few millimeters above a water bath with an IR temperature sensor abovethe glass slide to monitor the temperature variation during the mixingdose. With a waveform of 10% duty cycle, 150 mV input to the amplifier,and a 100 cycles per burst the temperature went from 25° C. to 60° C.within 30 seconds. Thereafter, the temperature maintained a steady-stateequilibrium condition. By modulating the acoustic wavetrain (e.g., dutycycle), the temperature was raised or lowered. Controlling thetemperature acoustically may be useful for accelerating stringencyprocesses for hybridizations. A small region of a device may be heatedwithout heating other regions. A target solution may be heated todenature it at the perimeter of an array without melting the hybridizedmolecules.

EXAMPLE NINETEEN Improved Signal From Mixing During Hybridization of aDNA Microarray, Closed Chamber

Mixing Ink Particles in a Closed Chamber.

A chamber was constructed as follows: A laminate consisting of pressuresensitive silicone transfer adhesive applied to the top and bottom of anOsmonics Poretics polyester membrane having a 5 micron pore size and a10 micron thickness, resulting in a total thickness of 60 microns wasdie-cut to form a chamber 21 mm square. The cut edge of the membrane wasexposed. A cover layer of cyclic-olefin (Zeon Chemicals Zeonex 1600),188 microns thick was punched to form two fill ports and applied to themembrane laminate to form a chamber. This chamber was bonded to astandard glass microscope slide. The volume of the chamber wasapproximately 40 microliters.

Ink was refined and prepared as above. The chamber was filled with amixture of the ink particles in 1×SSC. The ports were sealed with sealtabs (Grace Bio-Labs). The chamber was placed cover-side down at thesurface of the water bath in the sonic treatment system of FIG. 1.Acoustic energy was focused along the edges of the chamber such that thecut edge of the membrane was in the focal zone of the acoustic field. Atreatment waveform of 100 mV, 10 cycles per burst and 10% duty cycle wasapplied to the ultrasound transducer through a 55 dB RF amplifier tocreate an acoustic field focused on the slide. The slide was roboticallydithered in the horizontal plane during treatment so that focal zonemoved in a circular pattern relative to the edge of the array toaccommodate locational inaccuracies and to maximize the exposure of theedge of the membrane to the acoustic field.

The acoustic field applied to the edge of the laminate within thechamber resulted in circulating rotational flow of the fluid in “lobes”or eddies near the focal zone, as determined by visual observation. Thisflow pattern was similar to that of a “doublet” as is known in the fieldof fluid dynamics. As the focal zone was robotically moved around theedge of the chamber (while dithering), a border of mixed fluid occurredwithin the chamber. The treatment waveform described above resulted inborder zone of mixed fluid having a width of approximately 5 mm. Thevisually indicated border shows the region of primary circulation.Inside this border, the ink particles became organized into patternsformed by acoustic standing waves in the chamber and resisted movingwith any fluid flow that may have occurred.

Accelerating DNA Hybridization in a Closed Chamber.

A chamber similar to that described above was constructed and applied toa glass slide onto which was spotted a DNA probe array. The chamber wasfilled with a target solution containing cDNA molecules in ahybridization buffer. The slide was placed cover-side down at thesurface of the water bath in the sonic treatment system of FIG. 1 andtreated with a treatment waveform of 100 mV, 10 cycles per burst and a10% duty cycle for a period of 5 minutes at intervals of 30 minutes atan incubation temperature of 65° C. A similar slide was prepared andsubjected to the same conditions without the acoustic treatment. Afterthe treatment period was completed the slides were post-processed andscanned.

The slide receiving treatment showed substantially more fluorescentsignal than the untreated slide. There was no evidence that any of thespotted DNA probes had been removed or damaged by the treatment. Therewas no evidence of non-specific binding or cross-hybridization.

EXAMPLE TWENTY Improved Signal From Mixing During Hybridization of a DNAMicroarray, Nucleation Strip Applied Within a Closed Chamber

A strip of the laminate described in example 19, above, was bonded intoan otherwise standard chamber having dimensions of 12 mm×15 mm and athickness of 150 microns. The strip had a width of approximately 1 mmand a length of 8 mm. The chamber was then applied to a glass slide ontowhich an array of two distinct DNA oligonucleotide probes had beenspotted. A similar chamber without the strip of laminate was also placedon the same slide.

A target solution of oligonucleotides complimentary to those spotted onthe slide in a hybridization buffer was placed in both chambers. Theslide was placed upside down in the sonic treatment system of FIG. 1 atthe surface of the water bath at a temperature of 37° C. The chamberwith the strip of laminate was aligned with the focal zone of theultrasound transducer and treated for 15 minutes with a waveform of 10cycles of 125 mV, 10 cycles of 50 mV and an overall duty cycle of 10%.The slide was then post-processed and scanned with a slide scanner(Affymetrix #428).

The array in the treated chamber showed a substantially more uniformdistribution of fluorescent signal across the array than the chamberthat was not treated, by visual observation of the scanned image. Theoverall signal was higher too.

EXAMPLE TWENTY-ONE Mixing in a Chamber Using Unfocused Ultrasound

A chamber having an internal size of 22 mm square by 60 microns high wasplaced on a slide that was spotted with a reference grid of fluorescentspots and an array of two distinct oligonucleotide probes. The chamberwas filled with 30 microliters of a target solution in which the targetoligonucleotide molecules were complimentary to the spotted probes. Theslide and chamber were placed in a K&E Model 61-3128 ultrasonic pencleaner filled with degassed water at room temperature. The K&E pencleaner was operated a voltage approximately 60% of line voltage (with avariac) which corresponds to a minimum value that reliably causes mixingas determined by visual and auditory means. After 15 minutes oftreatment, the chamber was removed and the slide was post-processed andimaged.

The image showed that uniform mixing of the target solution occurred butthat cavitation damage (ablation) occurred to the spotted probes andfluorescent grid spots. The grid spots are quite durable and this is theonly condition that was observed to mechanically damage them throughouttesting with this type of slides.

EXAMPLE TWENTY-TWO Mixing Nucleation Patch Inserted into Chamber

A “nucleation patch” was bonded to a glass slide inside a standardhybridization chamber (Grace Bio-Labs #HBW1932) The nucleation patch wasin the configuration of a bandage in which a patch of membrane (OsmonicsPoretics 10 micron pore size, 20 micron thick) was held down by alaminate of 25 microns of pressure sensitive adhesive and a 37 micronplastic film. The dimensions of the membrane were 2 mm square and theoverall dimensions were 2 mm×6 mm.

The chamber was filled with refined white ink particles in 1×SSC andplaced chamber side down in the sonic treatment system of FIG. 1. Theslide was insonified with a 100 mV, 10 cycles per burst, 10% duty cyclewavetrain. When the focus was positioned over the center of thenucleation patch, a very strong flow (per visual observation) wasestablished from one side of the patch to the other, between themembrane and the glass slide. This “nucleation patch” could be insertedinto virtually any hybridization chamber. Further, because the flow isbetween the membrane and the slide, the flow will be independent of theinside height of the chamber. A variation would be to bond the patch tothe cover instead of the slide. Another variation would be to leave thetop film off so that the top of the patch bonds to the cover, preventingflow over the top of the patch. This would force the flow to follow alonger path and mix a larger area.

EXAMPLE TWENTY-THREE Beneficial Effect of Nucleating MembraneIncorporated into Chamber

Two chambers were constructed having an internal size of 9 mm×12 mm×150microns high. One chamber had Osmonics Poretics 5 micron pore size, 10micron thick polyester membrane incorporated into the laminate. Theother chamber had plain polyester film, 12 microns thick instead of themembrane. The chambers were laminated onto glass slides and filled with30 microliters of solution containing sub-micron white TiO2 particles in1×SSC.

The slides were treated in the system of FIG. 1. A series of treatmentswas applied to the edge of each chamber. The treatments had a voltage of40 mV to 120 mV, increasing in increments of 10 mV. The duty cycle was10% and there were 10 cycles in each burst. The presence of cavitationwas noted and the extent of the mixing from the edge was recorded foreach voltage in each chamber. The series was repeated twice for eachchamber. The extent of mixing was measured with digital calipers after15 seconds of treatment at each voltage.

The extent of mixing from the edge of the chamber in millimeters (mm) isshown in the following Table 2:

TABLE 2 Chamber Voltage Chamber without membrane with membrane (mV) 1strun 2nd run 1st run 2nd run 40 1.4 no mixing 2.5 1.3 50 2.1 no mixing2.7 2.1 60 1.4 1.6 3.1 .2.3 70 1.9 2.0 4.1 2.0 80 2.1 2.0 4.3 2.0 90 2.12.0 5.0 4.0 100  3.4 (sporadic) 4.3 5.5 4.3 110  4.8 (sporadic) 2.5 9.14.5 120  4.8 (sporadic) 3.5 (sporadic) 10.3  9.9

Mixing occurred in both chambers to some extent in response to theacoustic field. The chamber with the membrane showed much more extensivemixing, especially at higher voltages.

EXAMPLE TWENTY-FOUR Conduit Example

A chamber was constructed as follows: A laminate consisting of pressuresensitive silicone transfer adhesive applied to the top and bottom of anOsmonics Poretics polyester membrane having a 5 micron pore size and a10 micron thickness, resulting in a total thickness of 60 microns wasdie-cut to form a chamber 21 mm square. The cut edge of the membrane wasexposed. A cover layer of cyclic-olefin (Zeon Chemicals Zeonex 1600),188 microns thick was punched to form two fill ports and applied to themembrane laminate to form a chamber.

The chamber was modified as follows. A strip of thesilicone/membrane/silicone laminate measuring 1.5 mm by 6 mm wasattached to the inside of the cover layer parallel to one of the edgesso that a conduit 6 mm long was formed. The conduit had a height of 60microns and a width of approximately 1 mm. Both ends of the conduit werein free communication with the chamber. The chamber was bonded then to astandard glass microscope slide. The volume of the chamber wasapproximately 35 microliters.

The chamber was filled with a mixture of refined ink particles in 1×SSCand positioned in a Covaris E1 system. An acoustic waveform of 80 mVamplitude, 10% duty cycle and 10 cycles per burst was applied to thechamber. The chamber was positioned such that the focal zone wascentered on one end of the conduit.

Substantial convective flow was established in the conduit and in thechamber near the conduit. Flow rates were estimated to be severalmillimeters per second by visual observation.

Having described the invention, it is accordingly intended that allmatter contained in the above description be interpreted as illustrativerather than in a limiting sense. It is also intended that the followingclaims cover all of the generic and specific features of the inventionas described herein, and all statements of the scope of the inventionwhich, as a matter of language, might be said to fall therebetween.

1. An acoustic system, comprising: a vessel for holding one or moresamples, the vessel having a nucleation feature; an acoustic sourcespaced from and exterior to the vessel, the acoustic source forproviding focused acoustic energy in a frequency range of between about100 kHz and about 100 MHz and having a focal zone having a width of lessthan about 2 centimeters; a medium container for holding a liquid mediumfor transmitting acoustic energy from the acoustic source to the vessel;a vessel holder for holding the vessel, the vessel holder adapted toposition the vessel in the medium container at a location at leastpartially in the focal zone of the acoustic source; and a positioningsystem for controlling a relative position between the acoustic sourceand the vessel in the vessel holder, to selectively expose the vessel tothe focused acoustic energy, wherein exposure of the vessel having thenucleation feature to the focused acoustic energy imparts motion to oneor more samples in the vessel.
 2. The system of claim 1, furthercomprising a temperature control unit for controlling temperature of thesample.
 3. The system of claim 1, wherein the focused acoustic sourcegenerates a wavetrain substantially converging in a focal zone having awidth less than about 2 cm.
 4. The system of claim 3, wherein thefocused acoustic source for providing focused acoustic energy generatesa line focal zone having the width, a length and a depth.
 5. The systemof claim 4, wherein the line focal zone has a width of about 1.5 mm, alength of about 4 mm, and a depth of about 50 mm.
 6. The system of claim3, wherein the focused acoustic source generates a cigar-shaped focalzone of acoustic energy, and the width corresponds to a diameter of thecigar-shaped focal zone.
 7. The system of claim 6, wherein thecigar-shaped focal zone has a length that is greater than the width, andthe length is oriented in a vertical direction.
 8. The system of claim3, wherein the width corresponds to a diameter of the focal zone.
 9. Thesystem of claim 3, wherein the focused acoustic source generates awavetrain substantially converging in a focal zone having a width lessthan about 0.3 mm.
 10. The system of claim 3, wherein the focusedacoustic source energy generates an ellipsoidal focal zone.
 11. Thesystem of claim 10, wherein the width corresponds to a diameter of thefocal zone.
 12. The system of claim 1, wherein the focused acousticsource generates a wavetrain substantially converging in a focal zonehaving a width less than about 1 cm.
 13. The system of claim 1, whereinthe focused acoustic source generates a wavetrain substantiallyconverging in a focal zone having a width less than about 1 mm.
 14. Thesystem of claim 1, wherein the vessel is arranged to be sealed duringprocessing of the sample.
 15. The system of claim 14, comprising atemporary sealing layer for containing the sample within the vessel. 16.The system of claim 1, wherein the vessel is in a microtiter plateincluding a plurality of wells.
 17. The system of claim 1, wherein thevessel includes at least one of test tubes, centrifuge tubes, microfugetubes, ampoules, capsules, bottles, beakers, flasks, capillary tubes,pouches, bags, or vials.
 18. The system of claim 17, wherein the vesselis formed from a material including at least one of polyethylene,polypropylene, poly(ethylene teraphthlalate), polystyrene, acetal,silicone, polyvinyl chloride, phenolic, glass, metal,polyethylene/aluminum laminate, and polyethylene/polyester laminate. 19.The system of claim 1, wherein the focused acoustic source for providingfocused acoustic energy is modulated to produce multiple foci.
 20. Thesystem of claim 1, comprising a processor for controlling thepositioning system to selectively expose the vessel to the focusedacoustic energy.
 21. The system of claim 1, wherein the positioningsystem includes an extended work envelope for transferring the vessel.22. The system of claim 1, wherein the focused acoustic source generatesa wavetrain substantially converging in a focal zone having a width anda length, wherein the length is greater than the width.
 23. The systemof claim 22, wherein the focal zone has a depth that is greater than thewidth and the length.
 24. The system of claim 1, wherein the focusedacoustic source comprises a cylindrical segment.
 25. The system of claim1, wherein the focused acoustic source for providing focused acousticenergy generates a spot focal zone.
 26. The system of claim 1, whereinthe nucleation feature is carried by an element that is placed in thecorresponding reaction vessel and is unattached to the reaction vessel.27. The system of claim 1, wherein the element carrying the nucleationfeature includes a membrane or a microsphere.
 28. The system of claim27, wherein the element is a membrane that comprises micropores eachhaving a size ranging between about 5 microns and about 10 microns. 29.The system of claim 1, wherein the nucleation feature comprises acharacteristic size ranging between about 5 microns and about 10microns.
 30. The system of claim 1, wherein the nucleation featurecomprises a size and a geometry so as to promote formation of gascavities within a fluid, the gas cavities having a volume between about1 pL and about 3 mL.
 31. The system of claim 1, wherein the nucleationfeature comprises a size and a geometry so as to promote formation ofgas cavities within a fluid, the gas cavities having a volume betweenabout 10 nL and about 1000 nL.
 32. The system of claim 1, wherein thenucleation feature comprises a size that enables formation of at leastone bubble in the fluid, the bubble having a radius ranging betweenabout 0.27 microns and about 2.7 microns.
 33. The system of claim 1,wherein the vessel is one of a plurality of vessels.
 34. The system ofclaim 33, wherein each of the plurality of vessels is treated withfocused acoustic energy identically.
 35. The system of claim 33, whereinat least some of the plurality of vessels is treated with focusedacoustic energy differentially.
 36. The system of claim 33, whereinmultiple vessels in the plurality of vessels are selectively exposed tothe focused acoustic energy at the same time.
 37. The system of claim33, wherein each of the vessels in the plurality of vessels isindividually exposed to the focused acoustic energy.
 38. The system ofclaim 33, wherein each of the vessels in the plurality of vesselsincludes at least one nucleation feature.
 39. The system of claim 38,wherein the at least one nucleation feature includes at least one of apit, crevice, scratch, groove and ridge in a first surface.
 40. Thesystem of claim 1, further comprising: a liquid transmission mediumbetween the focused acoustic source and the vessel that transmits thefocused acoustic energy.
 41. An acoustic processing system, comprising:means for providing a focused acoustic field having a frequency range ofbetween about 100 kHz and about 100 MHz and having a focal zone having awidth of less than about 2 centimeters, the focused acoustic fieldoriginating from an acoustic source spaced from and exterior to avessel; means for holding a liquid medium in a medium container, theliquid medium for transmitting acoustic energy from the acoustic sourceto the vessel, and the vessel means having a nucleation feature; meansfor holding one or more samples in the vessel and holding the vessel ina vessel holder, the vessel holder adapted to position the vessel in themedium container at a location at least partially in the focal zone ofthe acoustic source; and means for selectively positioning thenucleation feature adapted to interact with said focused acoustic fieldto impart motion to fluid in the vessel means.